BURGER'S MEDICINAL

CHEMISTRY AND
DRUG DISCOVERY
Sixth Edition
Volume 4: Autocoids, Diagnostics, and
Drugs from New Biology

Edited by

Donald J. Abraham
Department of Medicinal Chemistry
School of Pharmacy
Virginia Commonwealth University
Richmond, Virginia

WILEY-
INTERSCIENCE
A John Wiley and Sons, Inc., Publication
 BURGER MEMORIAL EDITION
The Sixth Edition of Burger's Medicinal
Chemistry and Drug Discovery is being desig-
nated as a Memorial Edition. Professor Alfred
Burger was born in Vienna, Austria on Sep-
tember 6, 1905 and died on December 30,
2000. Dr. Burger received his Ph.D. from the
University of Vienna in 1928 and joined the
Drug Addiction Laboratory in the Department
of Chemistry at the University of Virginia in
1929. During his early years at UVA, he syn-
thesized fragments of the morphine molecule
in an attempt to find the analgesic pharma-
cophore. He joined the UVA chemistry faculty
in 1938 and served the department until his
retirement in 1970. The chemistry depart-
ment at UVA became the major academic
training ground for medicinal chemists be-
cause of Professor Burger.
Dr. Burger's research focused on analge-
sics, antidepressants, and chemotherapeutic
agents. He is one of the few academicians to
have a drug, designed and synthesized in his
vii
laboratories, brought to market [Parnate,
which is the brand name for tranylcypromine,
a monoamine oxidase (MAO) inhibitor]. Dr.
Burger was a visiting Professor at the Univer-
sity of Hawaii and lectured throughout the
world. He founded the Journal of Medicinal
Chemistry, Medicinal Chemistry Research,
and published the first major reference work
"Medicinal Chemistry" in two volumes in
1951. His last published work, a book, was
written at age 90 (Understanding Medica-
tions: What the Label Doesn't Tell You, June
1995). Dr. Burger received the Louis Pasteur
Medal of the Pasteur Institute and the Amer-
ican Chemical Society Smissman Award. Dr.
Burger played the violin and loved classical
music. He was married for 65 years to Frances
Page Burger, a genteel Virginia lady who al-
ways had a smile and an open house for the
Professor's graduate students and postdoc-
toral fellows.
 PREFACE
The Editors, Editorial Board Members, and
John Wiley and Sons have worked for three
and a half years to update the fifth edition of
Burger's Medicinal Chemistry and Drug Dis-
covery. The sixth edition has several new and
unique features. For the first time, there will
be an online version of this major reference
work. The online version will permit updating
and easy access. For the first time, all volumes
are structured entirely according to content
and published simultaneously. Our intention
was to provide a spectrum of fields that would
provide new or experienced medicinal chem-
ists, biologists, pharmacologists and molecu-
lar biologists entry to their subjects of interest
as well as provide a current and global per-
spective of drug design, and drug develop-
ment.
Our hope was to make this edition of
Burger the most comprehensive and useful
published to date. To accomplish this goal, we
expanded the content from 69 chapters (5 vol-
umes) by approximately 50% (to over 100
chapters in 6 volumes). We are greatly in debt
to the authors and editorial board members
participating in this revision of the major ref-
erence work in our field. Several new subject
areas have emerged since the fifth edition ap-
peared. Proteomics, genomics, bioinformatics,
combinatorial chemistry, high-throughput
screening, blood substitutes, allosteric effec-
tors as potential drugs, COX inhibitors, the
statins, and high-throughput pharmacology
are only a few. In addition to the new areas, we
have filled in gaps in the fifth edition by in-
cluding topics that were not covered. In the
sixth edition, we devote an entire subsection
of Volume 4 to cancer research; we have also
reviewed the major published Medicinal
Chemistry and Pharmacology texts to ensure
that we did not omit any major therapeutic
classes of drugs. An editorial board was consti-
tuted for the first time to also review and sug-
gest topics for inclusion. Their help was
greatly appreciated. The newest innovation in
this series will be the publication of an aca-
demic, "textbook-like" version titled, "Bur-
ger's Fundamentals of Medicinal Chemistry."
The academic text is to be published about a
year after this reference work appears. It will
also appear with soft cover. Appropriate and
key information will be extracted from the ma-
jor reference.
There are numerous colleagues, friends,
and associates to thank for their assistance.
First and foremost is Assistant Editor Dr.
John Andrako, Professor emeritus, Virginia
Commonwealth University, School of Phar-
macy. John and I met almost every Tuesday
for over three years to map out and execute
the game plan for the sixth edition. His contri-
bution to the sixth edition cannot be under-
stated. Ms. Susanne Steitz, Editorial Program
Coordinator at Wiley, tirelessly and meticu-
lously kept us on schedule. Her contribution
was also key in helping encourage authors to
return manuscripts and revisions so we could
publish the entire set at once. I would also like
to especially thank colleagues who attended
the QSAR Gordon Conference in 1999 for very
helpful suggestions, especially Roy Vaz, John
Mason, Yvonne Martin, John Block, and Hugo

Kubinyi. The editors are greatly indebted to
Professor Peter Ruenitz for preparing a tem-
plate chapter as a guide for all authors. My
secretary, Michelle Craighead, deserves spe-
cial thanks for helping contact authors and
reading the several thousand e-mails gener-
ated during the project. I also thank the com-
puter center at Virginia Commonwealth Uni-
versity for suspending rules on storage and
e-mail so that we might safely store all the
versions of the author's manuscripts where
they could be backed up daily. Last and not
least, I want to thank each and every author,
some of whom tackled two cha~ters.Their
contributions have provided our-field with a
sound foundation of information to build for
the future. We thank the many reviewers of
manuscripts whose critiques have greatly en-
hanced the presentation and content for the
sixth edition. Special thanks to Professors
Richard Glennon, William Soine, Richard
Westkaemper, Umesh Desai, Glen Kel-
logg, Brad Windle, Lemont Kier, Malgorzata
Preface
Dukat, Martin Safo, Jason Rife, Kevin Reyn-
olds, and John Andrako in our Department
of Medicinal Chemistry, School of Pharmacy,
Virginia Commonwealth University for sug-
gestions and special assistance in reviewing
manuscripts and text. Graduate student
Derek Cashman took able charge of our web
site, http://www.burgersmedchem.com, an-
other first for this reference work. I would es-
pecially like to thank my dean, Victor
Yanchick, and Virginia Commonwealth Uni-
versity for their support and encouragement.
Finally, I thank my wife Nancy who under-
stood the magnitude of this project and pro-
vided insight on how to set up our home office
as well as provide John Andrako and me
lunchtime menus where we often dreamed of
getting chapters completed in all areas we se-
lected. To everyone involved, many, many
thanks.
DONALD J. ABRAHAM
Midlothian, Virginia
 Dr. Alfred Burger

graph of Professor Burger followed by his comments to the American Chemical Society 26th Medicinal
listry Symposium on June 14, 1998. This was his last public appearance at a meeting of medicinal
ists. As general chair of the 1998 ACS Medicinal Chemistry Symposium, the editor invited Professor
?r to open the meeting. He was concerned that the young chemists would not know who he was and he
t have an attack due to his battle with Parkinson's disease. These fears never were realized and his
ients to the more than five hundred attendees drew a sustained standing ovation. The Professor was 93,
;was Mrs. Burger's 91st birthday.
 Opening Remarks

ACS 26th Medicinal Chemistry Symposium

June 14, 1998
Alfred Burger
University of Virginia

It has been 46 years since the third Medicinal Chemistry Symposium met at the University of
Virginia in Charlottesville in 1952. Today, the Virginia Commonwealth University welcomes
you and joins all of you in looking forward to an exciting program.

So many aspects of medicinal chemistry have changed in that half century that most of the
new data to be presented this week would have been unexpected and unbelievable had they
been mentioned in 1952. The upsurge in biochemical understandings of drug transport and
drug action has made rational drug design a reality in many therapeutic areas and has made
medicinal chemistry an independent science. We have our owri journal, the best in the world,
whose articles comprise all the innovations of medicinal researches. And if you look at the
announcements of job opportunities in the pharmaceutical industry as they appear in
Chemical & Engineering News, you will find in every issue more openings in medicinal
chemistry than in other fields of chemistry. Thus, we can feel the excitement of being part of
this medicinal tidal wave, which has also been fed by the expansion of the needed research
training provided by increasing numbers of universities.

The ultimate beneficiary of scientific advances in discovering new and better therapeutic
agents and understanding their modes of action is the patient. Physicians now can safely look
forward to new methods of treatment of hitherto untreatable conditions. To the medicinal
scientist all this has increased the pride of belonging to a profession which can offer predictable
intellectual rewards. Our symposium will be an integral part of these developments.
 CONTENTS

1 INSULIN AND HYPOGLYCEMIC 4 CHEMOKINE AND CYTOKINE

AGENTS, 1 MODULATORS, 129
Mark Sleevi Maria Elena Fuentes
Insmed Incorporated Tara Mirzadegan
Richmond, Virginia Robert S. Wilhelm
Roche Bioscience
Palo Alto, California
2 PEPTIDE AND PROTEIN
HORMONES, PEPTIDE
NEUROTRANSMITTERS, AND 5 COX-2 INHIBITORS AND
THERAPEUTIC AGENTS, 45 LEUKOTRIENE MODULATORS,
203
Victor J. Hruby
Catherine Gehrig de Chavez Randy L. Bell
Malcolm Kavarana Richard R. Harris
University of Arizona Andrew 0.Stewart
Department of Chemistry Abbott Laboratories
Tucson, Arizona Abbott Park, Illinois

3 INHIBITORS OF GASTRIC ACID 6 AGENTS ACTING ON

SECRETION, 85 PROSTANOID RECEPTORS, 265
Sonia Roberts David W. Jenkins
Iain M. McDonald Patrick P.A. Humphrey
James Black Foundation University of Cambridge
London, United Kingdom Glaxo Institute of Applied
Pharmacology
Cambridge, United Kingdom
Robert A. Coleman
Pharmagene Laboratories Ltd.
Royston, United Kingdom

xiii

7 RETINOIDS, 317
Mark Leid
Oregon State University
Laboratory of Molecular
Pharmacology
Department of Pharmaceutical
Sciences
College of Pharmacy
Corvallis, Oregon
8 VITAMINS, 359
John H. Block
Oregon State University
College of Pharmacy
Corvallis, Oregon
9 LIFESTYLE AND OVER-THE-
COUNTER DRUGS, 421
Khawla Abu-Izza
Sanofi-Synthelabo Research
Malvern, Pennsylvania
Vincent Li
Wyeth Consumer Healthcare
Richmond, Virginia
Graham Parr
Wyeth Consumer Healthcare
Hampshire, United Kingdom
10 RADIOPAQUES, 483
C. T. Peng
University of California
Department of Pharmaceutical
Chemistry
School of Pharmacy
San Francisco, California
and
San Jose State University
Nuclear Science Facility
San Jose, California
Contents
11 MICROARRAYS AND GENE
EXPRESSION PROFILING
APPLIED TO DRUG RESEARCH,
599
Brad Windle
Department of Medicinal Chemistry
Center for Bioelectronics, Biosensors
and Biochips (C3B)
Virginia Commonwealth University
Richmond, Virginia
Anthony Guiseppi-Elie
Department of Chemical
Engineering
Center for Bioelectronics, Biosensors
and Biochips (C3B)
Virginia Commonwealth University
Richmond, Virginia
12 SNPS: SINGLE NUCLEOTIDE
POLYMORPHISMS AND
PHARMACOGENOMICS-
INDlVIDUALLY DESIGNED
DRUG THERAPY, 617
Brian J. Puckett
MCV Campus
Virginia Commonwealth University
Richmond, Virginia
Steven G. Terra
JHM Health Science Center
University of Florida
Gainesville, Florida
Joe Walker
Orchid BioSciences
Princeton, New Jersey
13 PLASMID DNA-MEDIATED
GENE THERAPY, 651
Rajkumar Banerjee
Leaf Huang
Center for Pharmacogenetics
School of Pharmacy
University of Pittsburgh
Pittsburgh, Pennsylvania
INDEX, 679
 BURGER'S
MEDICINAL CHEMISTRY
AND
DRUG DISCOVERY
CHAPTER ONE

Insulin and Hypoglycemic

Agents
MARK SLEEVI
Insmed Incorporated
Richmond, Virginia

Contents
1 Introduction, 2
2 Current Drugs on the Market, 4
2.1 Insulin and Its Analogs, 4
2.1.1 Side Effects, Adverse Effects, 5
2.1.2 Absorption, Distribution, Metabolism,
and Elimination, 5
2.1.3 Physiology and Pharmacology, 8
2.1.4 Structure-Activity, 9
2.2 Insulinotropic Agents, 11
2.2.1 Side Effects, Adverse Effects, 15
2.2.2 Absorption, Distribution, Metabolism,
and Elimination, 15
2.2.3 Physiology and Pharmacology, 17
2.2.4 History, 18
2.2.5 Structure-Activity, 18
2.3 Insulin-Sensitizing Agents, 20
2.3.1 Biguanides, 21
2.3.1.1 Side Effects, Adverse Effects, 21
2.3.1.2 Absorption, Distribution,
Metabolism, and Elimination,
21
2.3.1.3 Physiology and Pharmacology, 23
2.3.1.4 Structure-Activity, 23
2.3.2 Thiazolidinediones, 24
2.3.2.1 Side Effects, Adverse Effects, 24
2.3.2.2 Absorption, Distribution,
Metabolism, and Elimination,
24
2.3.2.3 Physiology and Pharmacology,
24
2.3.2.4 History, 28
2.3.2.5 Structure-Activity, 28
2.4 a-Glucosidase Inhibitors, 31
Burger's Medicinal Chemistry and Drug Discovery 2.4.1 Side Effects, Adverse Effects, 31
Sixth Edition, Volume 4: Autocoids, Diagnostics, 2.4.2 Absorption, Distribution, Metabolism,
and Drugs from New Biology and Elimination, 31
Edited by Donald J. Abraham 2.4.3 Physiology and Pharmacology, 33
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc. 2.4.4 Structure-Activity, 34
1

1 INTRODUCTION
Insulin is a 58-kDa polypeptide hormone pro-
duced by p-cells in pancreatic islets of Langer-
hans regulating, in vivo, the storage, release,
and utilization of nutrient energy, carbohy-
drate in the form of glucose, fat, and protein,
in response to changing supply and demand.
Pancreatic p-cells respond to an increase in
circulating levels of glucose as occurs after in-
gestion of a meal by increasing the rate of in-
sulin release. The major metabolic actions of
insulin include the following: (1)promotion of
uptake and storage of glucose in liver and
muscle in the form of glycogen, (2) suppres-
sion of hepatic glycogenolysis and gluconeo-
genesis, (3)increasing the rate of glucose oxi-
dation in muscle, (4) suppression of lipolysis
and release of fatty acid from adipose tissue,
(5)enhancing triglyceride synthesis and stor-
age and de novo lipogenesis from carbohydrate
in liver and fat, and (6) promotion of amino
acid uptake and protein synthesis in muscle.
Thus, in normal individuals, the postprandial
increase in insulin suppresses the release and
utilization of nutrient energy substrate (glu-
cose, fatty acids, and amino acids) from he-
patic and muscle glycogen, fat depots, and
muscle protein, because these substrates are
temporarily plentiful due to digestion and ab-
sorption of the meal. Insulin also stimulates
the conversion of excess energy nutrient into
storage forms (glucose into glycogen, fatty ac-
ids into triglyceride, amino acids into protein)
and the sequestration of these in storage de-
pots for later use. In addition, elevated post-
prandial insulin increases oxidation of glucose
and decreases use of fatty acids in tissues (e.g.,
skeletal muscle) that can use either as meta-
bolic fuel. Under fasting conditions when in-
sulin levels are low, these processes are re-
versed, and metabolic fuel substrates are
released from storage and used to provide the
energy necessary for life processes.
Opposing the actions of insulin are the
counterregulatory hormones, glucagon, epi-
nephrine, growth hormone, and cortisol. In re-
sponse to low blood glucose, the counterregu-
latory hormones increase hepatic glucose
Insulin and Hypoglycemic Agents
output by stimulating glycogenolysis and glu-
coneogenesis and also inhibit glucose uptake
and utilization in peripheral tissue. This is
critical because, while most peripheral tissue
can utilize either glucose or fatty acids as fuel
for energy metabolism, the brain relies almost
exclusively on glucose, requiring 100-150
glday. For comparison, typical dietary intake
of carbohydrate is about 300-400 glday, and
total hepatic glycogen content is approxi-
mately 100 g. The brain has little capacity for
glucose storage, and hypoglycemia, depending
on degree and duration, can result in dizzi-
ness, seizures, coma, or death.
The term diabetes mellitus refers to a
group of disorders that are characterized by
hyperglycemia resulting from inadequate in-
sulin secretion, failure of insulin to elicit nor-
mal level of response in insulin sensitive tissue
(insulin resistance), or both. The vast majority
of cases of diabetes mellitus fall into two main
categories (1, 2), type 1 and type 2. In type 1
diabetes, which is also called insulin depen-
dent diabetes mellitus or IDDM, there is an
absolute deficiency in insulin secretion usu-
ally caused by autoimmune destruction of the
insulin producing p-cells. Type 2 diabetes, also
called non-insulin dependent diabetes melli-
tus or NIDDM, is characterized by both tissue
insulin resistance and an insulin secretory
deficit (3-5). Insulin resistance, particularly in
liver and muscle tissue, plays a major role in
the pathogenesis of type 2 diabetes. The ele-
vated fasting plasma glucose levels used to di-
agnose the disease result primarily from fail-
ure of insulin, often a t several times normal
concentrations, to adequately suppress he-
patic glucose output from gluconeogenesisand
glycogenolysis (3,6).Postprandial hyperglyce-
mia is caused by both impaired suppression by
insulin of hepatic glucose output and by the
diminished ability of insulin to stimulate glu-
cose uptake, storage and utilization, particu-
larly in skeletal muscle, which is the primary
site of postprandial glucose disposal (7). At the
cellular level, insulin stimulates several pro-
cesses required for glucose disposal including
glucose transport and phosphorylation, glyco-
gen synthesis, and glucose oxidation. Defects
1 Introduction
in the insulin activation of these processes are
present with insulin resistance in type 2 dia-
betes (3, 8-12). While the cause(s1 at the mo-
lecular level for the impairment in glucose dis-
posal are not completely understood, insulin
resistance is highly correlated with obesity.
There is good evidence supporting the hypoth-
esis that abnormalities in fatty acid metabo-
lism leading to elevated circulating free fatty
acids and to nonadipose tissue accumulation
of triglyceride and fatty acid metabolites con-
tribute significantly to insulin resistance (8,9,
13-16). Thus, insulin resistance is probably in
part a consequence of action of excessive fatty
acids and fatty acid metabolites on complex
signaling pathways and feedback loops that
regulate metabolic fuel utilization and stor-
age.
Insulin resistance is relatively common,
and it alone does not produce hyperglycemia.
Obesity, for instance, is almost always accom-
panied by some degree of insulin resistance,
but only a small proportion of obese individu-
als develop type 2 diabetes (8). This is because
a compensatory increase in circulating insulin
prevents hyperglycemia. In a population of
nondiabetic individuals with normal blood
glucose levels but a wide range of insulin sen-
sitivity, insulin secretion is inversely propor-
tional to insulin sensitivity and the product of
these two parameters is a constant (4,17-19).
Thus, hyperinsulinemia compensates for in-
sulin resistance and normal glycemic control
is maintained. Hyperglycemia occurs when
this compensation process fails, and further
decline in p-cell function is associated with the
progression of type 2 diabetes. Compensatory
hyperinsulinemia is the result of increased
p-cell mass (19,20), reduced insulin clearance
(19,21,22),and increased expression relative
to the normal glucokinase of a low K, p-cell
hexokinase, which catalyzes the first rate-lim-
iting step of glucose metabolism in the p-cell,
lowering the set point for insulin secretion in
response to glucose (19,21).In type 2 diabetes,
the insulin secretory deficit is generally be-
lieved to be due to both loss of p-cell mass and
insulin secretory function, but the underlying
causes are not well established. Chronic hy-
perglycemia (glucose toxicity) may be a factor
in reducing p-cell function as type 2 diabetes
becomes fully developed (4), and accumulation
of triglycerides and fatty acid metabolites (li-
potoxicity) in p-cells may also be important (4,
20).
Acute complications of hyperglycemia in-
clude thirst, polyuria, glucosuria, hunger, and
with severe elevations, hyperosmolar hyper-
glycemic nonketotic coma, an often fatal re-
sult of osmotic diuresis and dehydration. Pri-
marily in type 1 diabetes, ketoacidosis, caused
by a near total lack of insulin to suppress ex-
cessive lypolysis, hepatic oxidation of the re-
sulting fatty acids to acetoacetic and 3-hy-
droxybutyric acids, and accumulation of these
substances in circulation, is also potentially
fatal. Late complication with both type 1 and
type 2 diabetes are classified as microvascu-
lar-retinopathy, nephropathy and neuropa-
thy, and macrovascular-ischemic heart dis-
ease, peripheral vascular disease, and stroke.
Prevention of late complications presents the
greatest therapeutic challenge because it re-
quires near normalization of the elevated
blood glucose levels associated with the dis-
ease. Large randomized prospective clinical
trials have established that therapeutic inter-
vention to reduce hyperglycemia significantly
decreases microvascular complicationsin both
type 1 and type 2 diabetes (23).Although data
from large interventional trials focused on
postprandial glucose control are not available,
epidemiological studies show a clear associa-
tion between macrovascular risk and post-
prandial hyperglycemia (23,24).Management
of late complications of diabetes is a major
health care problem. Diabetic retinopathy is
the leading cause of blindness in the United
States, and diabetic nephropathy is responsi-
ble for one-third of all patients requiring kid-
ney transplants or dialysis (3, 25). Some
50,000 lower-extremity amputations per-
formed annually in diabetic individuals, re-
sulting from neuropathies and vascular insuf-
ficiency (26).
At the cellular and molecular level, hyper-
glycemia is believed to cause late complica-
tions in diabetes through a number of mecha-
nisms (27). First, glucose is a low affinity
substrate for aldose reductase, and hypergly-
cemia drives the reduction of glucose to sorbi-
to1 catalyzed by this enzyme. Excess flux
through this pathway results in cellular deple-
tion of reduced glutathione, rendering the cell
4 Insulin and Hypoglycemic Agents
susceptible to damage from oxidative stress.
Secondly, auto-oxidation of glucose is en-
hanced in hyperglycemia and results in the
formation of reactive dicarbonyl compounds
which in turn react with amino groups in pro-
teins to form covalently bonded adducts
termed advanced glycation end-products
(AGES).Modification of proteins alters their
function, both intracellularly and in the extra-
cellular matrix. Thirdly, hyperglycemia en-
hances glycolysis and increases intracellular
diacylglycerol through increased precursor
availability. Elevated diacylglycerol causes
abnormal stimulation of various isoforms of
protein kinase C, leading to decreased produc-
tion of nitric oxide and other abnormalities
affecting blood flow and capillary permeabil-
ity. Finally, hyperglycemia enhances the flux
of glucose through the hexosamine pathway
increasing production of glucosamine and its
derivatives. The hexosamine pathway is prob-
ably important in regulation of both carbo-
hydrate and fat metabolism, and increased
flux through this pathway may be an impor-
tant contributor to insulin resistance (28).
Through increased availability of cofactors, it
is also believed to lead to modification of tran-
scription factors and other proteins by
0-acetylglucosaminylation,altering both gene
expression and protein function. Inhibition of
endothelial nitric oxide synthase by this pro-
cess may be relevant to diabetic complications.
Each of these four pathogenic mechanisms
may reflect a single hyperglycemia-induced
process, overproduction of superoxide by the
mitochondrial electron transport chain (27).
The efficacy of therapeutic intervention in
the treatment of diabetes can be assessed by
monitoring plasma glucose levels in the fasted
state, but postprandial and bedtime measures
are also common. In normal individuals,
plasma glucose concentrations are maintained
within a narrow range of about 3.5-7.0 mM
throughout the day (29). Fasting or prepran-
dial plasma glucose concentrations are usually
less than 6.1 mM. After a meal, plasma glucose
levels rise and peak, usually within 30-60 min
and return to basal concentrations within 2-3
h. It is rare for 2-h postprandial glucose con-
centrations to exceed 7.8 mM in nondiabetic
individuals. For treatment regimens in both
type 1 and type 2 diabetes, the American Dia-
betes Association (ADA) recommends goals of
achieving average preprandial plasma glucose
concentrations of 5.0-7.2 mM, and 6.1-8.3
mMat bedtime (30). Guidelines of the Ameri-
can College of Endocrinology (ACE) recom-
mend targets of less than 6.1 mM for fasting
plasma glucose and 2-h postprandial blood
glucose concentrations of less than 7.8 mM
(23).The utility of measures of plasma glucose
in assessing the efficacy of treatment regi-
mens is limited in that these measures reflect
only the state of glycemia at the time of mea-
surement. Hemoglobin is nonenzymatically
glycosylated on the amino group of its termi-
nal valine at a rate proportional to the concen-
tration of glucose. Because 2-4 months are
required for complete turnover of hemoglobin,
determination of the fraction of hemoglobin
glycosylated in this way (HbA,, or AlC, usu-
ally expressed as percent) is used as a measure
for assessing glycemic control over time. In
normal individuals, HbA,, is in the range of
4.0-6.0%. The goal for treatment recom-
mended by the ADA is achievement of HbA,,
less than 7%, while the ACE targets less than
6.5%. The change in percent HbA,, is the gold
standard by which the efficacy of therapeutic
agents and treatment programs are assessed.
These therapeutic targets are difficult to
achieve. In a large U.S. epidemiological study,
mean HbA,, values for diabetic individuals
followed over a 10-year period and under a
variety of treatment regimens exceeded 9%
(31).
2 CURRENT DRUGS O N THE MARKET
Drugs currently available for treatment of hy-
perglycemia associated with diabetes mellitus
fall into four classes:
1. Insulin and its analogs
2. Insulinotropic agents
3. Insulin-sensitizing agents
4. a-Glucosidase inhibitors
2.1 Insulin and its Analogs
Insulin replacement therapy is the only effica-
cious treatment for type 1 diabetes and is also
useful for treatment of type 2 diabetes when
2 Current Drugs on the Market
b
-
Animal lnsulins Amino acid residues differing from human insulin:
porcine 830 Ala
bovine A8 Ala, A10 Val, 830 Ala
Insulin Analogs - Amino acid residues differing from human insulin:
lispro 828 Lys, 829 Pro
aspart 828 Asp
glargine A21 Gly, 831 Arg, 832 Arg
Figure 1.1. Amino acid sequence of human insulin and differences from the human sequence
-
for animal insulins and marketed insulin analogs.
poor p-cell function limits the efficacy of oral
medications. Insulin isolated from beef or
pork was used for many years to treat diabetes
mellitus. Animal insulins have largely been re-
placed by the human hormone, manufactured
by recombinant methods. Porcine insulin is
still available in the United States. Human in-
sulin is a 51 amino acid polypeptide and differs
from the porcine hormone by substitution of
one amino acid. It is composed of a 21 amino
acid A-chain and a 30 amino acid B-chain
joined together by a pair of disulfide linkages.
Three new insulin analogs with pharmacoki-
netic profiles considerably different than the
natural hormone have recently become avail-
able. Amino acid sequences for human and
porcine insulins along with marketed insulin
analogs are given in Fig. 1.1.
2.1 .I Side Effects, Adverse Effects. Hypo-
glycemia is the major and potentially very se-
rious side effect of therapeutic use of insulin
and its analogs. This is particularly true in
type 1 diabetes, where it is the major limiting
factor in glycemic management (32). The fre-
quency of severe hypoglycemia increases with
intensive insulin therapy designed to main-
tain near normal plasma glucose concentra-
tions. A meta-analysis of 14 clinical trials of at
least 6 months duration that included 1028
type 1 patients intensively treated with insu-
lin and 1039 patients treated with insulin in
less intense conventional regimens found that
intensive therapy increased the risk of a se-
vere hypoglycemic episode by approximately
threefold. A total of 846 of these patients suf-
fered at least one episode of severe hypoglyce-
mia (33). The incidence of severe hypoglyce-
mia is lower in type 2 diabetes, due to both
insulin resistance and less compromise of glu-
cose counterregulation. After 10 years of
treatment in the United Kingdom Prospective
Diabetes Study (UKPDS) in type 2 diabetics,
2.3% of the patients receiving insulin experi-
enced at least one hypoglycemic episode re-
quiring the help of a third-party or hospital-
ization (3).
When administered acutely by intravenous
infusion to type 1 diabetics, the newer insulin
analogs elicit responses very similar to insulin
with respect to hypoglycemia and hormonal
counterregulation (34, 35). When adminis-
tered subcutaneously in diabetes therapy, the
pharmacokinetic profiles of insulin analogs fa-
cilitate their use in treatment regimens de-
signed to closely mimic the natural variations
circulating insulin-the pancreatic response
to meals. As a consequence, some reduction in
the risk of hypoglycemia would be expected,
and this seems to be the case.
Insulin promotes the sequestration of fat,
and weight gain is a common side effect. This
is a concern particularly in type 2 diabetes in
which obesity is common. In the UKPDS,
obese patients on insulin therapy gained an
average of 4 kg more than those treated by diet
adjustment only.
2.1.2 Absorption, Distribution, Metabolism,
and Elimination. The plasma half-life of insu-
lin is quite short, 4-6 min, and the plasma
concentration of the hormone after subcuta-

neous administration is largely determined by
the rate of absorption from the subcutaneous
depot. Depending on concentration, the pres-
ence of certain divalent metal ions, especially
zinc, and pH, insulin may exist as a monomer
or self-associate in dimeric or hexameric struc-
tures. Zinc is usually found in insulin pharma-
ceutical formulations to encourage hexamer
formation and increase stability. Hexameric
zinc containing insulin formulations can be
prepared as solutions or as suspensions of
solid material obtained by crystallization, pre-
cipitation as an amorphous solid, or co-crystal-
lization with the basic protein protamine. So-
lutions have relatively rapid onset and short
duration, while the rate of absorption of insu-
lin from solid suspensions depends on the dis-
solution rate of the solid in the subcutaneous
depot. There are about 180 different formula-
tions of insulin marketed worldwide (29).
Properties of the most widely used formula-
tions are shown in Table 1.1.
Regular insulin (human) is clear solution of
zinc insulin. Its time of onset is 30-60 min
with peak activity at 2-3 hand duration of 3-6
h. The widely used insulin NPH (neutral prot-
amine Hagedorn) is a suspension of zinc insu-
lin co-crystallized with protamine and has a
slower onset (2-4 h) with longer time to peak
(4-10 h) and duration of action (10-16 h). In-
sulin lente is a suspension of mixed amor-
phous and crystalline zinc insulin, with onset
time of 3-4 h and a duration of 12-18 h. Insu-
lin ultralente, which is a suspension of crystal-
line insulin with a high zinc content, is the
slowest onset (6-10 h) and longest duration
(18-20 h) of these preparations. Peak plasma
concentrations occur after about 10 h with in-
sulin ultralente (36).
In recent years, results from large prospec-
tive clinical trials have shown the benefit of
maintaining near normal glycemia with inten-
sive insulin therapy in reducing diabetic late
complications. Ideally, insulin formulations
used in intensive therapy should be able to
mimic the normal variations in circulating in-
sulin levels. In nondiabetic individuals,
changes in insulin closely follow glucose, ris-
ing rapidly to a peak 30-45 min after inges-
tion of food and then rapidly returning to
basal levels within 2-3 h. Basal levels of insu-
lin are relatively stable and sufficient to sup-
Insulin and Hypoglycemic Agents
press excessive lipolysis and hepatic glucose
output without inducing hypoglycemia. The
pharmacokinetic profiles of human insulin
preparations are not ideal for use in dosing
regimens designed to provide constant basal
circulating levels of the hormone and rapid,
but relatively brief, increases associated with
meals. Regular insulin has too slow an onset
and too long a duration to match the natural
mealtime response, and peaks in insulin levels
with the longer duration preparations are un-
desirable in formulations providing the basal
component. The insulin analogs lispro and as-
part are more suitable than human insulin for
use in rapid onset, brief duration formula-
tions, while glargine is formulated for long du-
ration and is nearly peakless (Table 1.1).
With soluble regular insulin, the transport
of insulin hexamers from the subcutaneous
depot into the microcirculation is sterically re-
stricted, and a lag occurs as the hexamers
slowly dissociate on dilution in the interstitial
space into smaller species which can be ab-
sorbed (38). Insulins lispro and aspart are for-
mulated with zinc and also form hexamers,
but the slight amino acid sequence alterations
in analogs promote more rapid dissociation
and thus faster absorption. With insulin
glargine, which is intended to provide a pa-
tient's basal requirements, the modified
amino acid sequence of the peptide increases
the iso-electric point from pH 5.4 for human
insulin to 6.7. Because peptides are less solu-
ble at a pH near their iso-electric point,
glargine, which is formulated as a solution at
pH 4, forms a microprecipitates after subcuta-
neous injection as the pH increases to the
physiological level of 7.4. The slow dissolution
of these microprecipitates provides a rela-
tively constant delivery of insulin glargine
into the circulation over an extended -period of
time. Comparative time-course parameters of
the marketed insulin analogs in formulations
for subcutaneous injection are shown in Table
1.1.
Insulin is rapidly removed from circulation
and distributed into tissue by a process medi-
ated by its receptor, and thus all insulin sensi-
tive tissue take up the hormone (39). The pri-
mary site of clearance is the liver which
removes about half of portal insulin during
first-pass transit, but hepatic fractional ex-
 Table 1.1 Insulin Formulations for Subcutaneous Iqjection (37)
USP or
Nonproprietary Trade Peak Usual Effective Usual Maximum
Name Name Manufacturer Formulations Onset (h) Duration (h) Duration (h)
Insulin (human) Humulin Lilly R (Regular) 0.5-1 h
Novolin Novo Nordisk N (NPH) 2-4 h
v L (Lente) 3-4 h
U (Ultralente) 6-10 h
Insulin (porcine) Iletin I1 Lilly Regular 0.5-2 h
NPH 4-6h
Lente 4-6h
Lispro Humalog Lilly <15 min
Aspart Novolog Novo Nordisk 5-10 min
Glargine Lantus Aventis 1.1 h
8 Insulin and Hypoglycemic Agents
traction is controlled to some degree by a va-
riety of physiological factors. Absorption of
glucose in the intestinal tract increases he-
patic uptake of insulin, presumably mediated
by release of signaling factors from the intes-
tines since this does not occur when glucose is
administered by other routes. Free fatty acids
decrease insulin binding, degradation, and ac-
tion in the liver. On binding, the insulin-recep-
tor complex can dissociate and return insulin
into circulation or it can be internalized by
endocytosis. Most receptor bound insulin is in-
ternalized into endosomes and some insulin
signal transduction processes may occur
there. Endosomal insulin is either dissociated
from its receptor and returned to circulation
via retroendocytosis or partially degraded by
insulin-degrading enzyme (IDE) and the frag-
ments delivered to lysosomes for complete
degradation. Another enzyme, endosomal
acidic insulinase or EAI may also be important
in this process (40). The kidney is also a major
site for insulin clearance, removing about one-
half of the insulin in the peripheral circulation
by a receptor mediated process (39,411. Most
endosomal insulin in the kidney is delivered to
lysosomes where it is ultimately degraded. In-
sulin analogs, as well, are cleared and de-
graded in the kidney (41). On subcutaneous
administration, renal clearance of insulin is a
higher portion of the total than with the endo-
genously produced hormone because the nor-
mal process in which high concentrations of
newly secreted insulin are delivered directly
from the pancreas to the liver via the portal
circulation is bypassed. Renal failure may sig-
nificantly reduce insulin requirements.
2.1.3 Physiology and Pharmacology. Be-
fore the discovery and introduction of insulin
as a therapeutic agent, the only available
treatment for type 1 diabetes was a starvation
diet and the life expectancy for a child diag-
nosed with the disease was 1.3 years (42).
Judged against this standard, insulin therapy
for type 1 diabetes is hugely successful. Inten-
sive insulin therapy, usually three or more in-
sulin injections per day of regular insulin in
association with meals along with a longer act-
ing insulin to supply basal requirements or
continuous subcutaneous infusion both with
frequent monitoring of blood glucose to adjust
insulin dosage, dramatically decreases micro-
vascular complications. In the landmark
Diabetes Control and Complications Trial
(DCCT) intensive treatment reduced the risk
of retinopathy, nephropathy and neuropathy
by 35-90% compared with conventional ther-
apy of one or two insulin injections per day. A
median HbA,, level of 7.3% was obtained with
intensive therapy while that for the conven-
tionally treated group was 9.1% (43). How-
ever, the median HbA,, level for intensive in-
sulin therapy group shows that less than half
achieved the ADA goal for HbA,, in the rigor-
ous environment of a clinical trial, and by
5-years poststudy, the mean HbA,, for a sub-
set of the intensively treated cohort was 8.1%.
Insulin is commonly used in the treatment
of type 2 diabetes, but is most often introduced
when oral agents fail to adequately control hy-
perglycemia. A decrease in HbA,, of about 2%,
similar to that found with the most efficacious
oral agents, can be achieved with intensive in-
sulin therapy, although a lesser reduction is
probably the norm in general practice (3). Af-
ter 6 years in the UKPDS, insulin treated type
2 diabetics, newly diagnosed at the beginning
of the study, had lower fasting plasma glucose
levels, but similar HbA,, concentrations to pa-
tients treated with oral agents (44). The inva-
sive nature of subcutaneous administration,
greater weight gain and increased risk of hy-
poglycemia relative to oral agents may dis-
courage the early use of insulin. Therapy for
type 2 diabetes usually begins with lifestyle
interventions, particularly changes in diet and
exercise. In the UKPDS, only about 15% of
newly diagnosed patients were able to reach
target levels of glycemic control in 3 months of
intensive dietary therapy, and there was fur-
ther decline after l year (45). Oral agents are
introduced as monotherapy if lifestyle adjust-
ments are unsuccessful (46,47). Type 2 diabe-
tes is usually progressive with loss of p-cell
mass and function, and oral agents that stim-
ulate or rely on endogenous insulin produc-
tion become less efficacious over time. Glyce-
mic control with monotherapy in the UKPDS
deteriorated such that after 3 years, only
about one-half of the patients treated with a
single agent were able to achieve HbA,, less
than 7% (48). After 9 years of monotherapy,
only one-quarter were able to maintain this
2 Current Drugs on the Market
level of control, and the majority of patients
required multiple therapies. Ultimately insu-
lin is almost always required to achieve opti-
mal glycemic goals (46). Insulin can be intro-
duced in place of or in combination with oral
agents, but better results are often obtained
with combinations of insulin and oral agents
than with insulin alone (49-52).
The rapid onset, short acting insulin lispro
and aspart are used in treatment of both type
1 and type 2 diabetes in place of regular insu-
lin in intensive therapy regimens. The bene-
fits of these newer agents used by injection at
mealtime with optimized basal therapy in re-
duction of HbA,, in the rigorously controlled
environment of clinical trials are modest, 0.1-
0.4% when compared with regular insulin
(53). Reductions of up to 0.8% in HbA,, com-
pared with regular insulin have been obtained
with insulin lispro administered continuously
by infusion pump. When the long-acting insu-
lin glargine is used as the basal component of
insulin therapy, HbA,, levels are usually very
similar to those obtained with insulin NPH or
ultralente (531, although less weight gain in
type 2 diabetics and decreased frequency of
nocturnal hypoglycemia have been reported
with use of the analog. While the reductions in
HbA,, achieved with insulin analogs are mod-
est when compared with human insulin ther-
apy, even small improvements are believed to
reduce the risk of microvascular complications
in diabetes, and a survey of diabetic patients
suggests that a reduction in hypoglycemic ep-
isodes, better glycemic control, and improved
quality of life can often result from the use of
these agents (53,541.
At the cellular and molecular level, the
binding of insulin to a specific membrane
spanning receptor initiates a signal transduc-
tion cascade which ultimately produces the bi-
ological actions of the hormone. The insulin
receptor is a tetrameric protein comprised of
two a subunits that bind to insulin and two P
subunits that are linked by disulfide bonds
(55,561,and belongs to a subfamily of receptor
typrosine kinases which also includes the in-
sulin-like growth factor I (IGF-I) receptor.
The a subunits are extracellularly located,
while /3 subunits span the membrane and have
both intracellular and extracellular portions,
with the tyrosine kinase domain located intra-
cellularly in this subunit. Extracellular bind-
ing of insulin activates the receptor kinase ac-
tivity, and the receptor is autophosphorylated
on multiple intracellular /3 subunit tyrosine
residues (13,57). The insulin receptor shares
about 50% amino acid sequence homology
with the IGF-I receptor. Insulin and IGF-I
bind and activate both types of receptors, but
each has 2-3 orders of magnitude preference
for own receptor (58). There are two isoforms
of the insulin receptor differing by the pres-
ence or absence of 12 amino acids in the C-
terminus of the a subunit. The smaller iso-
form has about a two-fold higher affinity for
insulin than the longer, and this difference is
paralleled in sensitivity for metabolic actions
of insulin (59).
Ligand affinities for the insulin receptor
can be measured in binding assays based on
displacement of [1251]-labeledinsulin. The rel-
ative affinities for insulin and the insulin an-
alogs used clinically are shown in Table 1.2.
Many cell based assays can be used to assess
the functional potency of insulin and its ana-
logs. Primary adipocytes are exquisitely sensi-
tive to insulin and are often used for this pur-
pose. The relative potency of human insulin
and analogs in stimulating lipogenesis in pri-
mary rat adipocytes is also shown in Table 1.2.
The receptor affinities and functional poten-
cies of these analogs are similar to insulin,
with the exception that insulin glargine has
about a sixfold higher affinity for the IGF-I
receptor. To date, neither benefit nor risk has
been clearly demonstrated to be associated
with this higher affinity.
2.1.4 Structure-Activity. The structural ba-
sis and especially the role of specific amino
acid residues in the interaction of insulin mol-
ecules in forming dimers and hexamers or
binding to and activating insulin receptors
have been studied extensively by comparing
amino acid sequences from different species,
in structure-function studies using insulin an-
alogs, and with X-ray crystallography and mo-
lecular modeling. The insights gained have
been used primarily to design therapeutic
10 Insulin and Hypoglycemic Agents

Table 1.2 Relative Receptor Binding Affinities and Metabolic Potency for Human Insulin
and Analogs Used Clinically
Insulin Receptor IGF-I Receptor Lipogenesis
Affinity % AMinity % Potency %
[mean (SE)I [mean (SE)I [mean (SE)I
Human insulin 100 100 100
Lispro 84 (6) 156(16) 82 (3)
Aspart 92 (6) 81 (9) 101(2)
Glargine 86 (3) 641 (51) 60 (3)
- - - - -

Values are calculated as the ratio between EC,, for human insulin and EC,, for the analog. Binding affinities were
determined using solubilized human insulin and IGF-I receptors, employing TyrA14[12511-humaninsulin and Tyr31[1251]-
IGF-I as radioligands. Potency in lipogenesis is based on stimulation of incorporation of the label from D-[3Hl-glucoseinto
lipid in primary rat adipocytes. EC50 values for human insulin are 0.01 and 200 nM for insulin and IGF-I receptor binding
respectively, and 45 pM in lipogenesis (60).

agents with improved pharmacokinetic pro-
files,but which mimic the natural hormone in
other ways as closely as possible.
Insulin forms both dimers and hexarners,
and dimerization occurs mainly through inter-
actions of certain B-chain residues (Fig. 1.1),
involving amino acids at positions B8, B9, B12,
B13, B16, and B23-28 (61, 62). Hexamer for-
mation is stabilized mainly through coordina-
tion of the six HisBlO residues (one from each
monomer) to two zinc ions, but burial of a non-
polar surface made up by amino acids at posi-
tions A13, A14, B1, B2, B14, B17, and B18 also
contributes (62, 63). The amino acid residues
that are considered most essential for high
binding affmity of insulin to its receptor are at
positions A1-A3, A16, A19, A21, B6, B12, B15,
B23-B25, and perhaps A13 and B17. Minor
contributions are made by residues at A8, B9-
10, B13, and B16 (64, 65). Substitutions for
residues at B26-30 are often possible while
maintaining high affinity for the insulin re-
ceptor, but mainly effect IGF-I receptor
binding.
X-ray crystallography has yet to provide de-
tailed information at the atomic level for insu-
lin bound to its receptor, due to difficulties in
obtaining X-ray crystal structures of mem-
brane-spanning ligand-receptor complexes.
The three-dimensional structure of the com-
plexed receptor has been obtained with a com-
bination of electron cryomicroscopy using
gold-labeled insulin to locate the binding do-
main and high resolution structures of insulin
and individual subdomains of the receptor (66,
67). In this structure, insulin binds by bridg-
ing from a binding site on one extracellular a
subunit to a dissimilar site in the second a
subunit of the receptor dimer. Insulin resi-
dues at positions A4, A8, A17, A.21, and B29
interact with polar amino acids in one a sub-
unit primarily through electrostatic salt
bridges, while there are both hydrophobic and
polar interactions between insulin residues at
positions B9, B10, B12, B13, B16, B17, B21,
B22, B24, B26, A5, and A15 and the other cu
subunit. There is only a modest correlation
between insulin amino acid residues found to
contribute significantly to binding af!inity
based on structure-activity studies and those
which closely interact with the receptor in the
three dimensional structure. Not all close con-
tacts observed in the structure necessarily
make large contributions to binding energy,
and factors such as change in conformation
induced by substitution of residues not di-
rectly interacting with the receptor may also
contribute to changes in binding affinity.
Recombinant methods have enabled the
preparation of a very large number of insulin
analogs, but relatively few have been exten-
sively characterized pharmacologically. A sin-
gle point mutation in the proinsulin gene
identified as cause of a case of familial hyper-
proinsulinemia led to the synthesis of
[AspBlOI-insulin(68-70)and some related in-
sulin analogs. Lacking the HisBlO residue
which stabilizes hexamer formation through
Zn2+ binding, [AspBlO-insulin is absorbed
about twice as rapidly as regular insulin. It
exhibits a 3.5-fold increased binding affhity
for the insulin receptor and is about twice as
potent as insulin in metabolic assays. Surpris-
ingly, [AspBlOI-insulinis 10- to 20-fold more
2 Current Drugs on the Market
potent than the parent in mitogenic assays
(71,72).While [AspBlOI-insulinhas increased
affinity for the IGF-I receptor relative to insu-
lin; its relative affinity for this receptor is
about 1000-fold lower than for the insulin re-
ceptor, and enhanced mitogenic potency has
been observed in a cell line lacking IGF-I re-
ceptors. The mitogenic/metabolic potency ra-
tios of [AspBlOI-insulin and several other in-
sulin analogs are inversely correlated with
their insulin receptor dissociation rate con-
stants (71), suggesting that increased mito-
genic potency results primarily from sus-
tained activation of the insulin receptor
tyrosine kinase through formation of long-
lived ligand-receptor complexes. A dose-
dependent increase in incidence of adenocar-
cinomas in laboratory animals treated with
suprapharmacological doses of [AspBlOI-
insulin prevented development of this analog
as a therapeutic agent (65).Thus, the pharma-
cologic actions produced on administration of
a particular insulin analog may depend not
only on insulin receptor affinity, intrinsic ac-
tivity and selectivity, but also half-life of the
ligand-receptor complex.
Modifications of the native insulin se-
quence at the C-terminus in the B26 to B30
region have led to all of the marketed insulin
analogs. This region is not critical for binding
to the insulin receptor (65, 731, but sequence
modifications here often yield analogs with al-
tered tendencies to form aggregates. A sys-
tematic investigation (61) of amino acid sub-
stitutions in the insulin B-chain with the
intent of reducing dimer stability by introduc-
ing charge-charge or steric repulsion led to re-
placement of ProB28 with aspartic acid to af-
ford [AspB28]-insulin(insulin aspart). Insulin
aspart retains the biological profile of insulin,
but is more rapidly absorbed. Synthesis of a
series of analogs having LysB29 replaced with
proline and varying the amino acid at B28 led
to identification of [LysB28 ProB291-insulin
(insulin lispro) as a fully potent insulin analog
with decreased association in solution and
faster onset of action than the parent (38,74).
Sequence modifications which increase the
isoelectric point of insulin from 5.4 toward
neutrality led to analogs which form micropre-
cipitates and have extended duration of action
(29, 75). Following this concept, addition of
-
two basic arginine residues to the C-terminus
of insulin and substitution of glycine for
AspA21 gave the long acting [GlyA21ArgB31
ArgB321-insulin (insulin glargine) with an iso-
electric point near pH7.0 (29, 76). Long acting
insulin analogs also result from fatty acid ac-
ylation of the side-chain amino group of
LysB29, increasing size and promoting bind-
ing of these analogs to albumin, thus delaying
absorption from the subcutaneous injection
site (29, 77, 78). [LysB29-tetradecanoyl des-
B301-insulin (insulin detemir) in which albu-
min binding is further enhanced by deletion of
the terminal threonine at B30 is undergoing
clinical evaluation.
2.2 lnsulinotropic Agents
Insulinotropic agents directly stimulate the
release of insulin from pancreatic p-cells, and
are thus useful only in the treatment of
NIDDM. The drugs in this class are often di-
vided into the subclasses of sulfonylureas and
glinides. These compounds are structurally re-
lated and share a common mechanism of ac-
tion, and are described together.
Sulfonylureas have been used as hypogly-
cemic agents since the mid-1950s. They act by
closing membrane-bound ATP-sensitive po-
tassium (KATp)channels on the p-cell, causing
depolarization and the opening of voltage-
gated calcium channels. The resulting influx
of Ca2+ triggers exocytosis of insulin. The
structures and properties of the major mar-
keted sulfonylureas are shown in Tablel.3,
roughly in order of increasing potency. Com-
pounds in the first generation of this class
such as chlorpropamide, tolbutamide, and to-
lazamide are still in use, but are less potent
than the more recently introduced second-
generation drugs like glipizide, glyburide, and
glimepiride. Treatment regimens with sulfo-
nylureas provide more or less continuous hy-
poglycemic activity with once- or twice-daily
dosing.
As with the rapid onset, short-duration in-
sulin~,two short-acting glinide insulin secre-
tagogues, repaglinide and nateglinide (Table
1.4), have recently become available. Taken
immediately before meals, these agents pro-
vide increased insulin secretion in the post-
prandial period. Like sulfonylureas, the com-
pounds act by closing pancreatic p-cell KATp
Table 1.4 Structures and Properties of Insulinotropic Agents: Glinides
USP or
Nonproprietary
Name Trade Name Manufacturer Chemical Class Structure Dose t m t,,, Durationa
Repaglinide Prandin Novo Nordisk Benzoic Acid CH3 0 0.- mg 1h 1-1.4 h 4-6 h
11 2, 3, or 4 x daily

Nateglinide Starlix . Novartis Phenylalanine 120 mg

3 x daily

"Ref. 3.
2 Current Drugs on the Market
channels, but have rapid onset after oral ad-
ministration and short duration of action. Re-
paglinide is the more potent of the two, but
nateglinide reportedly has somewhat faster
onset and shorter duration of action (81).
2.2.1 Side Effects, Adverse Effects. Sulfonyl-
ureas are generally well tolerated. The major
safety concern is severe hypoglycemia, with
the longer-acting agents carrying a greater
risk. In the United Kingdom Prospective Dia-
betes Study (UKPDS), the proportion of pa-
tients experiencing major hypoglycemic epi-
sodes was 1.0 and 1.4% per year with
chlorpropamide and glyburide, respectively,
as compared to 0.7% with diet and 1.8% with
insulin therapy (82). Sulfonylureas are for the
most part subject to hepatic metabolism,
yielding less active or inactive metabolites
that are then eliminated through the kidney.
Patients with impaired hepatic or renal func-
tion risk severe hypoglycemia because of accu-
mulation of active drug in circulation. As with
insulin, gain in body weight is common. The
University Group Diabetes Program (UGDP)
study found an increased risk of cardiovascu-
lar mortality associated with the treatment of
type 2 diabetes with tolbutamide, although
the methods used have been criticized. The
UKPDS showed no increase in cardiac events
with sulfonylurea treatment (3, 83). Less is
known on long-term side effects with the
newer glinide short-acting insulinotropic
agents. Like sulfonylureas, weight gain is a
side effect of repaglinide or nateglinide ther-
apy. In year-long preapproval clinical trials
with repaglinide, 13%of patients discontinued
the use of the drug because of adverse events,
most commonly hyperglycemia or hypoglyce-
mia. In studies of 6 months or longer with
nateglinide, 0.3% of patients discontinued be-
cause of hypoglycemia.
2.2.2 Absorption, Distribution, Metabolism,
and Elimination. All of the marketed sulfonyl-
urea are nearly completely absorbed, and the
class has excellent oral bioavailability. A
quantitative structure-activity relationship
(QSAR) model for human drug oral bioavail-
ability (84) has recently been developed, in
which the descriptors A log D (log D,,, - log
D,,4) and log D,,, both proved to be important
contributors in addition to a variety of struc-
tural descriptors. The log D,., descriptor is the
log of the octanol-water distribution coeffi-
cient at pH 6.5 and a measure of lipophilicity
at the pH of the small intestine. The A log D
term is the difference between log D measured
at pH 6.5 and that measured at pH 7.4, the pH
of blood. It has a positive value for acids and is
negative for bases. The optimum value for log
D,,, is about -0.3 and the A log D weighting
coefficient is positive, indicating that, all
things being equal, there is better bioavailabil-
ity for acids than for neutral or basic com-
pounds. An interpretation of the A log D out-
come is that a higher fraction of un-ionized
compound at pH 6.5 aids absorption, whereas
a higher fraction of ionized compound at pH
7.4 aids in preventing first-pass metabolism.
Sulfonylureas are weak acids, with pK, values
in the range of 5.0 to 6.3 (85), and values (84)
for log D,., for glipizide, glyburide, and tolbu-
tamide (1.31,1.85, and 1.11, respectively) pre-
dict excellent oral bioavailability in the ab-
sence of metabolically labile functionality.
Although the marketed sulfonylureas are
all well absorbed, they differ in the time re-
quired to reach maximum blood levels (Table
1.3) and in metabolic fate and in rate and
mode of elimination. These differences can
have important implications for safe use of the
various drugs. Accumulation of these agents
in vivo can lead to extended episodes of serious
or fatal hypoglycemia through overstimula-
tion of insulin release. Insulinotropic drugs,
for instance. which are metabolized to active
compounds that are eliminated solely by renal
excretion, pose a serious risk of hypoglycemia
in renally compromised individuals.
The volumes of distribution for the various
sulfonylureas are similar, with V, values in
the range of 0.1-0.3 Lkg, indicating limited
distribution beyond extracellular water (86).
They are highly bound to serum protein
(90-99%).
The older first-generation sulfonylureas
are extensively metabolized and primarily ex-
creted renally. Tolbutamide is transformed by
oxidation of the benzylic methyl group, yield-
inga hydroxymethyl metabolite, (la),which is
further oxidized to the corresponding carbox-
ylic acid, (lb). These metabolites have little
activity.

(la) R = CH20H
(lb) R = COOH
Tolazamide is more slowly eliminated than
tolbutamide, with a half-life of about 7 h. Like
tolbutamide, it is metabolized (87) by oxida-
tion of the benzylic methyl group to a hydroxy-
methyl metabolite, which has some activity,
and to the corresponding carboxylic acid. It is
also hydroxylated on the 4 position of the
hexahydroazepine ring and hydrolyzed to p-
toluenesulfonamide. The metabolites are ex-
creted largely in the urine along with about
7%of the parent drug. Gliclazide is somewhat
more slowly absorbed and has a longer half-
life (10-12 h) than that of tolbutamide and
tolazamide. Excretion is 60-70%in urine and
10-20%in feces. It is also metabolized (88) by -
oxidation of the benzylic methyl group to a
hydroxymethyl compound and a carboxylic
acid, which are the main metabolites in urine.
Lesser amounts of five other metabolites,. hy- -
droxylated at various positions on the azabicy-
clooctyl ring, have also been identified. The
metabolites are not believed to have signifi-
cant hypoglycemic activity (86). Less than 1%
of the drug is found unchanged in urine.
Acetohexamide is metabolized by reduction
in the liver of its ketone function to an alcohol.
hydroxyhexamide, which is pharmacologically
active (79, 86). After oral administration, the
plasma concentration of the parent compound
peaks in 1-2 h, them falls rapidly. The active
metabolite has a longer half-life and prolongs
the duration of action. Hydroxyhexamide is
excreted primarily in the urine with a small
amount of the parent and other clinically un-
important metabolites. Chlorpropamide is
quite long lived in circulation, with an elimi-
nation half-life of about 36 h. It is slowly me-
tabolized by hydroxylation at the 2 or 3 posi-
tion of the propyl substituent, yielding
metabolites that retain some activity and by
N-dealkylation to p-chlorobenzenesulfonyl-
urea (86, 89). Urinary excretion is the main
Insulin and Hypoglycemic Agents
route of elimination from the system. About
20%of the dose is excreted as unchanged drug.
With the newer second-generation com-
pounds, glyburide is metabolized by hydroxy-
lation on the cyclohexane ring, yielding
4-trans and 3-cis-hydroxy derivatives. Both of
these metabolites may contribute to the over-
all hypoglycemic activity (90). An elimination
half-life of 15 h, on the basis of an improved
analytical method, is probably more in line
with the observed duration of action (91).Gly-
buride has been reported to accumulate in
pancreatic islets, a finding'that was unique to
this drug (92). It is eliminated as its metabo-
lites with approximately equal amounts in
urine and bile. Glipizide is similarly metabo-
lized primarily by hydroxylation at the 3 and 4
positions of the cyclohexane ring, although the
metabolites are not believed to contribute to
the activity of the drug. It is eliminated some-
what more rapidly than either glyburide or
glimepiride, mainly in the urine as hydroxy-
lated metabolites and polar conjugates. Of the
second-generation agents, glipizide has the
most rapid onset, but also has a somewhat
shorter duration than that of either glyburide
or glimepiride.
Glimepiride is completely metabolized by
oxidation of the pendant methyl substituent
on the cyclohexane ring to a hydroxymethyl
metabolite (2a)and a carboxylic acid (2b).The
hydroxymethyl metabolite has about one-
third of the hypoglycemic activity of the par-
ent in an animal model, whereas the other was
not active. Glimepiride is eliminated with a
half-life of about 9 h, and metabolites, but no
parent drug, are found in both urine (60%)
and feces (40%),with (2b) the predominant
species in urine and (2a) in feces.
The short-acting insulinotropic agents re-
paglinide and nateglinide are rapidly and com-
pletely absorbed. After oral administration,
both drugs reach peak plasma levels in about
1 h. Steady-state volumes of distribution (V,,)
after intravenous (i.v.) injection are 31 L for
repaglinide and 10 L nateglinide, indicating
little distribution beyond the plasma volume
for the latter and a somewhat wider distribu-
tion for the former. Both drugs are >98%
bound to serum protein.
Repaglinide is extensively metabolized in
the liver by dealkylation and oxidation to a
2 Current Drugs on the Market
(2a) R = CHzOH
(2b) R = COOH
dicarboxylic acid, (3a),by further dealkylation
of (3a) to amine, (3b),and by formation of an
acyl glucuronide of the parent. Elimination is
rapid, with a half-life of about 1 h. About 90%
of drug-related species are excreted in the fe-
ces in radiolabeled experiments, mainly as
(3a) with less than 2%of the parent (93).
Nateglinide is metabolized by the mixed-
function oxidase system primarily by oxida-
tion of the isopropyl side chain. It is eliminated
mainly in the urine (94), primarily as (4a)
(4a) R1 = H, Rz = OH
(4b) R1 = OH, Rz = H
(4c) R1 = OH, Rz = OH
(33%) and unchanged parent (16%), along
with lesser amounts of (4b)and (4c),acyl gluc-
uronides, and dehydration or dehydrogena-
tion product (5). Metabolite (5) is approxi-
mately equipotent with nateglinide, but only
small amounts are formed.
2.2.3 Physiology and Pharmacology. Sulfo-
nylureas and the carboxylic acid glinide hypo-
glycemic agents stimulate insulin secretion by
a direct action on pancreatic islet p-cells, and
are a mainstay in treatment of type 2 diabetes
in patients with good p-cell function. The
pharmacologic actions of these drugs are
largely, if not entirely, mediated by increased
insulin production, and thus are essentially
the same as insulin. Sulfonylurea therapy typ-
ically has been found to reduce fasting hyper-
glycemia by 3.3-3.9 mmol/L and HbA,, by
1.5-2% (3). There is less clinical experience
with the short-acting glinides, but repaglinide
appears to have a similar effect on HbA,, and
the very short acting nateglinide somewhat
less as monotherapy.
Both sulfonylureas and glinides exert their
action by binding to and closing an ATP-sen-
sitive potassium channel (KAT,) in the plasma
membrane of the p-cell (95, 96). Glucose is
transported into these cells predominately by
the non-insulin-dependent GLUT2 trans-
porter, and the rate of glucose transport into
the cell and metabolism reflect plasma glucose
concentration. At low glucose concentrations,
the transmembrane potential of pancreatic
p-cells is maintained at about -70 mV by an
outward flow of K' ions through the KATp
channel. After a rise in plasma glucose, the
increase in glucose metabolism leads to a rise
in the ATP/ADP ratio and closure of KATp

Table 1.5 Binding Parameters for
Sulfonylureas and Glinides with HEK
EBNA[Human SURl] Cell Membranes
Ki Hill Coefficient
nM (SEM) (SEW
Glyburide 2.3 (0.4) 1.0 (0.05)
Glimepiride 4.5 (0.9) 1.1(0.13)
Glipizide 100 (24) 0.82 (0.12)
Repaglinide 240 (38) 0.90 (0.07)
Nateglinide 1300 (110) 0.80 (0.05)
Tolbutamide 270,000 (17,000) 1.05 (0.09)
channels, thus depolarizing the cell. The
change in membrane potential results in the
opening of voltage-gated Ca2+ channels and
an increase in intracellular Ca2+,which trig-
gers insulin release. Sulfonylureas, by block-
ingpotassium current through the KATpchan-
nel, produce the same effect.
The KATpchannel is composed of two pro-
tein subunits in a ratio of 4:4. One subunit,
termed Kir6.2, is a member of the inward rec-
tifying potassium channel family. The other
regulatory subunit, SUR1, belongs to the ABC
(ATP-binding cassette)-transporter super-
family. Sulfonylureas bind with the KATp
channel at both a low aMinity site on Kir6.2
and a high affinity site on SUR1, which con-
fers the channel-blocking activity. The sulfo-
nylurea binding site appears to be located on
the intracellular side of SUR1. The number of
bound sulfonylurea molecules required for
KATpchannel closure is not known. Because
there are four SURl subunits in each KATp
channel, binding ratios greater than unity
may be required to block potassium current.
The relative potency of various sulfonyl-
ureas and glinides determined by direct com-
petitive displacement of [ 3H]glyburide from
membrane preparations of recombinant hu-
man embryonic kidney cells expressing high
levels of SURl is shown in Table 1.5 (97). Both
sulfonylureas and the glinides displace r3H]-
glyburide from SUR1-containing membrane
preparations. Moreover, they appear to oc-
cupy very similar sites on the receptor protein,
given that neither repaglinide nor nateglinide
has any effect on the dissociation kinetics of
[3H]glyburide.
The high affinity binding of sulfonylureas
to SURl suggests the existence of an endoge-
Insulin and Hypoglycemic Agents
nous ligand. Two polypeptides isolated from
porcine brain, a- and P-endosulfine, have been
found to inhibit sulfonylurea binding to
SUR1, although the functional significance is
not clear (98, 99).
2.2.4 History. As early as 1942, Janbon
(100) observed a high incidence of hypoglyce-
mia in typhoid patients treated with a bacte-
riostatic isopropylthiadiazole derivative of
sulfanilamide. Subsequently, during antibac-
terial clinical testing of N-(4-aminobenzene-
sulfony1)-N'-butylurea, carbutamide (6) was
also found to have similar hypoglycemic activ-
ity. Structure-activity studies at Boehringer
Mannheim and at Hoechst led to the introduc-
tion of carbutamide and tolbutamide as antidi-
abetic agents in 1956.
2.2.5 Structure-Activity. The more re-
cently introduced insulinotropic agents are
complex, richly functionalized molecules, the
optimized products of many extensive syn-
thetic and pharmacological investigations
(101-117) of structure-activity relationships
(SAR).Common features found in potent com-
pounds (102, 103, 106) of this class are sum-
marized in Fig. 1.2, illustrated by the use of
glyburide, repaglinide, and nateglinide as ref-
erence structures. The relationship to other
compounds in Table 1.3 is readily apparent.
Hypoglycemic sulfonylureas and glinides
contain an acidic functional group (A in Fig.
1.2) that is required for insulinotropic activity.
In all of the marketed drugs of this class, the
acidic group is attached to a phenyl ring (B in
Fig. 1.2). The acidic group is generally a sulfo-
nylurea, a propionate, or carboxylate, al-
though compounds containing other acidic
moieties like sulfonylsemicarbazides, sulfo-
nylaminopyrimidines, sulfonylcyanoguani-
dines, and sulfonamidonitroethylenes are
quite active.
Substitution on the acidic function with a
pendant lipophilic group (C in Fig. 1.2) greatly
enhances (103) affinity for the SURl and in-
creases selectivity for SURl over related
SUR2A receptors found in heart and skeletal
muscle and SUR2B receptors found in smooth
muscle. In the earliest sulfonylureas, this sub-
2 Current Drugs on the Market
Amido Linker (D)
Aromatic or
Heterocyclic
Tail with ortho
Repaglinide
Figure 1.2. Common structural features found in sulfonylurea, benzoic acid, and phenylalanine
derived hypoglycemic agents.
stituent is often an N-propyl or N-butyl group,
whereas cycloalkyl groups are most common
in later compounds. The pendant lipophilic
group cannot be attached to benzoic acid de-
rivatives like repaglinide, but it has been pro-
posed that the alkyl group of the 2-ethoxy sub-
stituent on the phenyl ring (C) of this
compound occupies a similar site on the recep-
tor (106). Phenylalanine derivatives like
nateglinide have a chiral center adjacent to
the carboxylate. In these compounds, the R
Phenyl Ring Acidic Head
(B)
I
1 1 Pendant
CH3 Nateglinide
configuration at this center is required for ac-
tivity (104, 105).
The acidic group in these agents is attached
to a phenyl ring (C), which is most often'sub-
stituted para to the acidic function. In first-
generation sulfonylureas, the para-substitu-
ents are small groups like methyl, acetyl, or
chloro. Introduction of larger groups com-
posed of an amido linker (D) attached to an
aromatic or heterocyclic tail (E) greatly in-
creases the potency of the second-generation
20
compounds. In the amido linker, the carbon
and nitrogen atoms of an amide are incorpo-
rated into a four-atom chain, with the amido
nitrogen occupying the third position from the
phenyl ring (C). In the sulfonylureas gly-
buride, glipizide, and glimepiride, the carbon
atom of the amido carbonyl group is a t the
fourth position. Some benzoic acid derivatives
like meglitinide (7), which has tolbutamide-
like potency (107, 110, 1171, have a similar
arrangement of the amido linker. The ar-
rangement in which the carbonyl is at the sec-
ond position as in repaglinide or the sulfonyl-
urea (8) [active at 0.25 mg/kg p.0. in rabbits
(11611 also affords highly potent compounds.
In this alternative, alkyl substitution of the
carbon at the fourth position improves po-
tency and activity resides in the S-enantio-
mers in both sulfonylurea and benzoic acid
derivatives. Within the amido linker, the car-
bony1 group may be more important than the
amide NH, and it has been suggested that each
alternative chain arrangement positions its
respective 0x0 function to accept a hydrogen
bond from the same donor group in the SURl
protein (106).
Insulin and Hypoglycemic Agents
Nateglinide lacks an equivalent to the
amido linker (Din Fig. 1.2) and the aromatic
or heterocyclic tail (E). In related N-benzoyl-
phenylalanine compounds (102), the SAR de-
termined for substitution on phenyl ring C
parallels that for sulfonylureas, and com-
pound (9), containing a side chain somewhat
like those found in the second-generation sul-
fonylureas and repaglinide, was active ([3Hl-
glyburide binding inhibition IC,, = 28 fl;
inhibition of KATpchannel activity EC,, = 3.5
fl).
The amido linker found in second-genera-
tion sulfonylureas and repaglinide terminates
in an attachment to an aromatic or heterocy-
clic group (E in Fig. 1.21, which is often substi-
tuted ortho to the point of attachment. Sub-
stituents such as alkyleneimino, alkoxy, or 0x0
having an oxygen or nitrogen atom adjacent to
the ring afford potent compounds (106). Glipi-
zide lacks this ortho substituent, but the
pyrazino nitrogen may serve a similar func-
tion in binding.
Conformational analysis of some second-
generation sulfonylureas and repaglinide
identified low energy conformations of these
agents, in which the pharmacophoric groups
derived by SAR studies could be best superim-
posed (106). The proposed binding conforma-
tions for glyburide and repaglinide are shown
in Fig. 1.3.
2.3 Insulin-Sensitizing Agents
Two subclasses of insulin-sensitizing agents
are currently available, the biguanides and the
thiazolidinediones. They are described sepa-
rately in this section because drugs in these
2 Current Drugs on the Market
Figure 1.3. Proposed binding conformations (106)
for glyburide and repaglinide based on the selection
of calculated low energy conformers, which allow
approximate superimposition of the major pharma-
cophoric groups.
subclasses do not share a common mechanism
of action and are not structurally similar.
2.3.1 Biguanides. First introduced in the
late 1950, metformin (Table 1.6) is best de-
scribed as an antihyperglycemic agent because
it does not normally cause hypoglycemia. It
enhances insulin sensitivity and is not effec-
tive in the absence of insulin (118). Metformin
lowers blood glucose levels in NIDDM patients
by suppressing hepatic glucose output and en-
hancing peripheral glucose uptake, but the
underlying mechanism(s) is not completely
understood. Other biguanides produce similar
effects, but concern over potential to cause lac-
tic acidosis has largely led to their removal
from the market.
2.3.1.1 Side Effects, Adverse Effects. The
major side effects associated with metformin
therapy are gastrointestinal, including diar-
rhea, nausea, abdominal discomfort, and an-
orexia, which improve with dose reduction
and can be minimized by slow dose titration
(3, 118). Lactic acidosis, a serious often fatal
side effect associated with biguanides, is rare
with metformin, with incidence estimated at
0.01 to 0.08 per 1000 patient-years. With met-
formin therapy, lactic acidosis most often oc-
curs in patients with renal insufficiency, prob-
lems with alcohol abuse, or liver and
cardiopulmonary disease (3, 118, 119). Met-
formin is removed from the system almost ex-
"
elusively by renal elimination of the un-
changed drug. Thus, poor renal function may
lead to accumulation. Hypoglycemia is rare
with metformin monotherapy.
2.3.1.2 Absorption, Distribution, Metabo-
lism, and Elimination. Biguanides are strongly
basic, and metformin (pK, = 11.5) is com-
pletely protonated and cationic over the phys-
iologic pH range. Absorption of metformin
from the GI tract after oral administration is
incomplete, with 20 to 30% recovered in the
feces (120). In single oral dosing studies, a lack
of dose proportionality indicates decreased ab-
sorption with increasing dose. Peak blood lev-
els are obtained in 2 to 4 h. The average vol-
ume of distribution after a single oral dose of
850 mg is 654 L. The pharmacokinetics of met-
formin have been described by use of a two-
compartment model (120), with rapid distri-
bution to a central plasma compartment
followed by slow transfer to a deep compart-
ment. Tissue concentrations higher than that
in plasma are found in liver, kidney, salivary
glands, and the intestinal wall. Metformin
does not bind to plasma proteins, but does par-
tition into erythrocytes. It is not metabolized
and is excreted unchanged in the urine. After
i.v. administration, renal clearance is about
3.5 times creatinine clearance, an indication of
tubular secretion.
Metformin is slowly and incompletely ab-
sorbed and rapidly eliminated without hepatic
metabolism. This pharmacokinetic profile
may make drug accumulation and lactic acido-
sis less likely
" to occur with metformin than
with other biguanides. Sales of the longer-act-
ing biguanide phenformin (lo),for instance,
which is metabolized in the liver by aromatic
Table 1.6 Structure and Properties of Metformin
USP or
Nonproprietary Chemical
Name Trade Name Manufacturer Class Structure Daily Dose 4"- t,, Durationa
Metformin Glucophage Bristol-Meyers Biguanide 1500-2550mg 2 4h 6.2 h > 3 4 wk
Squibb
H3C,
N
I
CH3
"Ref. 3.
2 Current Drugs on the Market
hydroxylation, were discontinued in the
United States in 1977 because of its associa-
tion with lactic acidosis.
2.3.1.3 Physiology and Pharmacology. Used
in treatment of type 2 diabetes, metformin has
typically been found to reduce fasting hyper-
glycemia by 1 to 3.9 mmolIL and HbA,, by 1 to
2% (3, 119, 121, 122), approximately equally
efficacious with sulfonylureas, and also to re-
duce postprandial hyperglycemia. As mono-
therapy, it generally does not induce hypogly-
cemia nor does it increase insulin secretion. It
does, however, require the presence of insulin.
In vivo, the principal antidiabetic effect of
metformin is suppression of hepatic glucose
output. This could, in theory, arise from either
inhibition of glycogenolysis or gluconeogene-
sis or both, and studies examining the relative
contribution of each have produced conflicting
results (123, 124). A recent study employing
both 13C-NMR and isotope techniques con-
cluded that suppression of endogenous glu-
cose production with 3 months of metformin
monotherapy in type 2 diabetics was com-
pletely attributed to a reduction in gluconeo-
genesis, although hepatic glycogen content did
increase (125). Metformin enhances insulin
sensitivity and glucose disposal in peripheral
tissue (3, 118). At the cellular level, this ap-
pears to be attributable in part to increased
glucose transport (119, 125-129). Metformin
treatment is often associated with an im-
proved plasma lipid profile (121, 1301, lower-
ing plasma triglyceride and LDL cholesterol.
At the molecular level, precise target recep-
tor(~) have not been identified. Metformin has
been reported to activate the AMP-activated
protein kinase system (AMPK) in primary
hepatocytes (131). AMPK has been proposed
as a key regulatory enzyme of carbohydrate
and fat metabolism (132). This kinase is acti-
vated through an allosteric mechanism by
binding 5'-AMP and also by phosphorylation.
Activation of AMPK by phosphorylation is 5'-
AMP dependent because the binding of AMP
to the enzyme makes it a better substrate for
AMPK kinase, which phosphorylates AMPK,
and a poorer substrate for protein phospha-
tase 2C, which dephosphorylates it. AMPK ki-
nase is also activated by 5'-AMP. These ac-
tions of 5'-AMP are antagonized by high
concentrations of ATP, and thus the system
responds to the AMPIATP ratio. Metformin
has no direct effect on the activity of partially
purified AMPK, but in primary hepatocytes it
increases the phosphorylation and activity of
AMPK in a time-dependent manner. This may
occur through modulation of AMPK kinase or
PP2C, or through a change in the AMPIATP
ratio. At relatively high concentrations, met-
formin inhibits respiratory chain complex I in
intact hepatocytes through an unknown sig-
naling pathway (133), but not in isolated mi-
tochondria. Such fnhibition might lead to an
increase in the AMPIATP ratio and thus
account for increased phosphorylation of
AMPK. In any case, activation of AMPK would
be expected to increase fatty acid oxidation
and glucose uptake in muscle, while decreas-
ing hepatic gluconeogenesis and lipogenesis, a
pattern of activity that is similar to that ob-
served with metformin.
In Xenopus oocytes (126, 134), treatment
with metformin has been shown to increase
insulin receptor tyrosine kinase activity asso-
ciated with activation of phospholipase C and
increased levels of IP, and intracellular Ca2'.
Thus metformin, through increasing receptor
tyrosine kinase activity, may facilitate IRS
protein phosphorylation and activation of
phosphatidylinositol-3-kinase, leading to in-
creased glucose uptake.
2.3.1.4 Structure-Activity. The available
data on the relationship of structure to hypo-
glycemic activity for substituted biguanides
result from a very limited number of studies,
generally in healthy animals (135-137). Alkyl
substitution (structure 11)at R, increases ac-
tivity through n-pentyl, but decreases with
longer chain length, branching, or with cyclic
alkyl substituents. When R, is aralkyl, phen-
ethyl is the most potent. Potency is highest
with a hydrogen or methyl substituent at R,.

2.3.2 Thiazolidinediones. The thiazolidine-
diones (Table 1.7) are the most recently intro-
duced class of oral agents for treatment of type
2 diabetes, improving insulin sensitivity and
lowering blood glucose, free fatty acid, and tri-
glyceride levels. The thiazolidinediones are
PPARy (peroxisome proliferator-activated re-
ceptor y) agonists. The PPARy receptor is a
member of the nuclear hormone receptor fam-
ily of ligand-activated transcription factors
that regulates gene expression of several
genes involved in fatty acid and carbohydrate
metabolism and adipocyte differentiation
(138).
2.3.2. I Side Effects, Adverse Effects. Be-
cause of their recent introduction, long-term
side effects are less well known than with the
other classes of hypoglycemic agents. Both
rosiglitazone and pioglitazone are generally
well tolerated. Weight gain and minor edema
are associated with thiazolidinedione therapy,
and their use in patients with moderate to se-
vere chronic heart failure is not advised. De-
creases in hemoglobin levels and hemocrit
have also been found. The first marketed thia-
zolidinedione, troglitazone (12), was with-
drawn because of increased risk of hepatotox-
icity. Approximately 1.9% of patients treated
with troglitazone in preapproval controlled
clinical trials showed an increase in plasma of
the liver enzyme alanine aminotransferase
(ALT) greater than three times the upper
limit of the normal range. In trials with ros-
iglitizone or pioglitizone, the incidence of sig-
nificantly elevated ALT was low and similar to
that of placebo. However, regular monitoring
of liver enzymes is recommended when these
drugs are used (139,140).
Insulin and Hypoglycemic Agents
2.3.2.2 Absorption, Distribution, Metabo-
lism, and Elimination. Rosiglitazone is well ab-
sorbed, with absolute bioavailability of 99%.
After oral dosing, peak blood levels are ob-
served in about 1 h. Rosiglitazone is highly
bound to plasma protein (99.8%), with a
steady-state volume of distribution of appoxi-
mately 17.6 L. Pioglitazone is also well ab-
sorbed, reaching maximum blood levels in
about 2 h. Binding to plasma proteins is
greater than 99%,with a single dose volume of
distribution of 0.63 Lkg.
Rosiglitazone is metabolized in the liver
primarily by N-demethylation and by hy-
droxylation of the pyridine ring at the 5 posi-
tion (para to the side chain), followed by con-
jugation (141, 1421, but the metabolites are
not believed to contribute significantly to clin-
ical effect. The drug is eliminated, with a half-
life of 3 to 4 h, primarily in the urine (64%),
with lesser amounts in the feces (23%).Piogli-
tazone is extensively metabolized (143, 144)
by 0-dealkylation to (13), by benzylic oxida-
tions of methylene groups adjacent to the pyri-
dine ring (14a, 14b, 14c, and 14e) and by o
oxidation of the pyridine ethyl substituent
(14d).
Metabolites (14a), (14b), and (14c) have
hypoglycemic and hypolipidemic effects in
Wistar fatty rats, and may contribute signifi-
cantly to the pharmacological activity of pio-
glitazone in humans. Serum half-life of the
parent drug is 3-7 h, whereas that for total
pioglitazone-related species is 16-24 h. From
15 to 30% of an oral dose is excreted in the
urine, primarily as metabolites and their con-
jugates. The balance is presumably excreted in
the bile.
2.3.2.3 Physiology and Pharmacology. Ro-
siglitazone and pioglitazone have only re-
cently been introduced for the treatment of
type 2 diabetes, and evaluations of the efficacy
of these drugs over years of therapy are not yet
available. In large preapproval clinical trials of
24-26 weeks in type 2 diabetics (1451, piogli-
tazone treatment (45 mg, once daily) as mono-
therapy reduced fasting blood glucose by 3.6
and 3.8 mmol/L compared with placebo and
HbA,, by 1.5 and 1.6%, respectively. In two
similar 26-week preapproval studies (1461,
rosiglitazone monotherapy (4 mg, twice daily)
resulted in decreases in mean fasting plasma
 Table 1.7 Structures and Properties of Thiazolidinediones
USP or
Nonproprietary Trade Daily
Name Name Manufacturer Chemical Class Structure Dose Lax t,, Durationa
Rosiglitazone Avandia SmithKline Thiazolidinedione 4-8 mg 1-1.75 hr 3-4 hr 23-4 wk
Beecham
CH3
I
VI
h) f NyN-O

Pioglitazone Actos TakedaILilly Thiazolidinedione

"Ref. 3.

glucose levels of 3.4 and 4.2 mmoVL relative to
a placebo-treated group, with a mean decrease
of 1.5% in HbA,, in both. In a 52-week gly-
buride-controlled study, rosiglitazone at 2 or 4
mg twice daily was initially less efficacious
than glyburide in reducing hyperglycemia, but
by the end of the study, both drugs produced a
similar response (change in HbA,,: -0.7%
with glyburide and -0.5% with rosiglitazone).
This might suggest that the hypoglycemic ac-
tivity of rosiglitazone and perhaps other thia-
zolidinediones, although initially less than
that of sulfonylureas, is more durable over
time. In addition to hypoglycemic activity,
thiazolidinediones reduce insulin levels and
improve insulin resistance, markedly reduce
plasma free fatty acids, increase the storage of
fat, and often improve the blood lipid profile
(139. 147). The thiazolidinediones also in-
crease adipose tissue cell differentiation. Ros-
iglitazone promotes the differentiation of
human subcutaneous preadipocytes into adi-
pocytes, but has little effect on preadipocytes
from visceral adipose tissue depots (147, 148).
As discussed later in this section, the effects of
thiazolidinediones on carbohydrate metabo-
lism are probably at least in part a result of the
action of these compounds on the storage and
metabolism of fat.
Thiazolidinediones bind to and activate
peroxisome proliferator-activated receptor y
(PPARy) (149-152). PPARy is a member of
the PPAR family of nuclear receptors, which
are ligand-activated transcription factors reg-
ulating storage and metabolism of fatty acids.
The three members of the PPAR family,
termed a, 6, and y, are activated by fatty acids
and fatty acid metabolites. PPARa is the tar-
get receptor for the fibrate class of hypolipi-
demic agents. Activation of this receptor in
rodents, but not humans, leads to an increase
in the number and size of hepatic peroxi-
somes, organelles involved in P-oxidation of
fatty acids, a property unique to PPARa, from
which the name of the receptor family was de-
rived. Less is known about the function of
PPARS, although a PPARG-selective agonist
has been reported to increase reverse choles-
terol transport (153).
PPARy and transcription factors of the
CCAATIenhancer-binding protein family (C/
EBPa, -P, and -6) are integral members of the
Insulin and Hypoglycemic Agents
genetic cascade leading to adipogenesis (1541,
the process of adipocyte differentiation. Acti-
vation of PPARy, which is highly expressed in
adipose tissue (155-159), leads to upregula-
tion of the expression of genes regulating fatty
acid transport, storage, and oxidation. There
are two isoforms of PPARy, termed PPARyl
and PPARy2, differingin that PPARy2 has 28
additional amino acids at the N-terminus in
humans (157).Both PPARyl and PPARy2 are
abundant adipose tissue, with mRNA for
PPARy2 generally about 10 to 15% of that of
PPARyl. Lesser amounts of PPARy2 have
been found in liver, whereas PPARyl appears
to be more widely distributed (152, 156, 158-
160).PPARy activation also leads to decreased
expression of gene-encoding enzymes re-
quired for gluconeogenesis in the liver and
suppressing glucose oxidation in muscle (150,
161).
Comprehensive mRNA profiling has been
used to identify genes regulated directly or in-
directly by PPARy in various tissues in Zucker
diabetic fatty rats (161). Proteins for which
PPARy activation leads to increased mRNA in
white adipose tissue include enzymes acetyl-
CoA carboxylase, fatty acid synthase, and
dihydrolipoamide acyltransferase [a compo-
nent of the pyruvate dehydrogenase (PDH)
complex] required for lipogenesis, and phos-
phoenolpyruvate carboxykinase (PEPCK),
glycerol-3-phosphate acyltransferase, and
long-chain acyl-CoA synthetase for triglycer-
ide biosynthesis. In muscle, expression of
pyruvate dehydrogenase kinase 4 (PDK4) is
decreased. PDK4 phosporylates and deacti-
vates PDH complex, limiting oxidative glucose
disposal. Thus, suppression of PDK4 gene ex-
pression would lead to an increase in muscle
glucose oxidation. In contrast to adipose tis-
sue, liver expression of PEPCK is suppressed
by PPARy activation along with other
enzymes required for gluconeogenesis, includ-
ing pyruvate carboxylase and glucose-6-
phosphatase.
In common with other PPARs, PPARy
forms an obligate heterodimer (152,162) with
9-cis retinoic acid receptors (RXR) and regu-
lates gene expression through binding of the
heterodimer to DNA response elements, con-
taining two copies of the sequence AGGTCA,
separated by a single nucleotide (DR1 motif).
2 Current Drugs on the Market
Specificity is likely a result of small differences
in optimal DNA-binding sites and differences
in tissue distribution (150). Agonist binding
allows recruitment of coactivator proteins
such as CAMPresponse element binding pro-
tein (CBP) or steroid receptor coactivator 1
(SRC-I), which mediate the transcriptional
activation of target genes. Coactivator pro-
teins contain one or more leucine-rich LxxLL
motifs known as nuclear receptor (NR) boxes,
which bind to hydrophobic pockets formed on
agonist binding to PPARy and other nuclear
receptor proteins. The nature of the xx amino
acid residues as well as residues flanking the
NR box appears to determine the coactivator-
receptor specificity (163).
The affinity of particular agents for binding
to PPARy can be assessed with conventional
binding assays (149) employing radioligands
such as [3H]rosiglitazone.A scintillation prox-
imity assay (164) amenable to high through-
put screening has also been developed. Varia-
tions of functional transactivation assays are
used that measure expression of PPARy tar-
get genes in mammalian cells transfected with
both a chimeric receptor expression construct,
composed of the ligand-binding domain of
PPARy coupled with the DNA-binding do-
main of the yeast transcription factor GAL4,
and a construct containing a GAL4 response
element and reporter gene combination (151,
152, 165). Reporter genes such as chloram-
phenicol acetyltransferase (1651, secreted pla-
cental alkaline phosphatase (SPAP) (1511, or
firefly luciferase (149) whose products can be
conveniently assayed have been used. Because
GAL4 is not found in mammalian cells, activa-
tion of other nuclear receptors does not inter-
fere with these assays. Comparative binding
affmities (152) and PPARy agonist activity
(151) in a PPARy-GAL4ISPAP assay for tro-
glitazone, pioglitazone, and rosiglitazone are
shown in Table 1.8. These compounds have no
significant activity in PPARa- or PPARS-spe-
cific assays, and the relative potencies of these
thiazolidinedione agents in PPARy assays
compares well with in vivo hypoglycemic po-
tency in humans.
PPARy has a significant role in adipocyte
differentiation and in the regulation of fat
storage and utilization. However, thiazol-
idinediones are used elinically as hypoglyce-
Table 1.8 Thiazolidinedione PPARy
Binding Affinity and Cell-Based PPARy
Agonist Activity
PPARy Human PPARy
Binding Agonist Activity
Potency (nM) ECm (a)
Troglitazone 7900 0.55
Pioglitazone 5500 0.58
Rosiglitazone 40-200 0.043
mic agents and to improve insulin sensitivity.
The mechanism(s) by which PPARy activa-
tion produces these clinically useful effects is
not completely established. Although at lower
levels than those in adipose tissue, PPARy is
present in skeletal muscle and liver, tissues
primarily responsible for glucose disposal and
gluconeogenesis. Hence, direct regulation by
PPARy of expression of key enzymes control-
ling these processes cannot be ruled out. On
the basis of comprehensive mRNA profiling, it
has been estimated that administration of a
PPARy agonist in a diabetic rodent model al-
tered the expression level directly or indirectly
of approximately 10% of all genes in white or
brown adipose tissue, whereas about 2% of the
genes in liver and 1% in skeletal muscle were
regulated (161). The alteration in specific en-
zyme levels predicted by the observed changes
in mRNA in liver and muscle (for instance,
PEPCK and PDK4) should lead to suppression
of hepatic gluconeogenesis and increased glu-
cose oxidation in muscle. The administration
of the PPARy agonist troglitazone to mice en-
gineered to lack adipose tissue improved insu-
lin sensitivity (166), and preincubation with
troglitazone increased glucose uptake, glyco-
gen synthase fractional velocity, and fatty acid
oxidation in cultured human myotubes (167).
Thus, PPARy agonists like the thiazol-
idinediones may have a direct action on mus-
cle and liver that is independent of their action
on fat.
Alternatively, improved insulin sensitivity
and lowered blood glucose with these agents in
treatment of NIDDM may be an indirect effect
of their action on adipose tissue. Signaling
through a number of adipocyte-derived fac-
tors, including TNF-a (168),leptin (169-1721,
resistin (172) and adiponectin (1731, is altered
by PPARy activation in ways that could lead to

enhanced insulin sensitivity. In addition, free
fatty acids, triglycerides, and products of fatty
acid metabolism regulate insulin sensitivity.
An inverse relationship between insulin sensi-
tivity and fasting plasma free fatty acid con-
centrations has been observed in offspring of
type 2 diabetic parents (8). Increased plasma
concentrations of free fatty acids contribute to
insulin resistance by inhibition of glucose up-
take, glycogen synthesis, and glucose oxida-
tion in muscle (8, 9, 16, 174) through regula-
tory interaction of fatty acid metabolites with
components of the insulin signal transduction
cascade and enzymes controlling glucose
metabolism.
In normal individuals, skeletal muscle has
the capacity to switch from the predominant
uptake and oxidation of fatty acid as metabolic
fuel under fasting conditions to greater up-
take, storage, and oxidation of glucose under
insulin stimulation. In obesity, which is linked
strongly with type 2 diabetes, metabolic in-
flexibility in the form of lower fasting rates of
lipid oxidation and failure of insulin to sup-
press fatty acid uptake and utilization is be-
lieved to lead to triglyceride accumulation in
muscle (9, 175). The amount of triglyceride in
skeletal muscle is significantly associated with
insulin resistance. Thiazolidinediones pro-
mote the storage of fat and also redistribution
of intracellular triglyceride from insulin-re-
sponsive organs into adipose tissue (1751, ac-
counting, perhaps, for improved insulin sensi-
tivity with the use of these drugs. Although
increasing the fat store might intuitively seem
more likely to increase than to decrease insu-
lin resistance, this is thought to be depot spe-
cific. Visceral adipose tissue volume, rather
than general obesity or subcutaneous adipose
tissue volume, appears to correlate with insu-
lin resistance (147). After 6 months of rosigli-
tazone therapy, a modest but significant in-
crease in subcutaneous fat mass was observed,
with no change in visceral fat.
Elevated free fatty acids and intracellular
accumulation of triglycerides in pancreatic
p-cells may also play a role in loss of p-cell
mass and insulin secretory function, which oc-
cur in NIDDM as the disease progresses (176,
177). In short-term studies, rosiglitazone pre-
vented loss of p-cell mass in Zucker Diabetic
Insulin and Hypoglycemic Agents
Fatty rats (1781, suggesting utility in slowing
or preventing the progression of diabetes.
2.3.2.4 History. In the course of investiga-
tions on the fibrate class of hypolipidemic
agents at Takeda (179, 1801, a series of 5-
(4-alkoxybenzy1)-2,4-thiazolidinedioneswere
shown to reduce insulin resistance in geneti-
cally diabetic and obese animals. Ciglitazone
(IS),which became the prototype for this class
of drug, was found to reduce hyperglycemia,
hyperlipidemia, and hypertriglyceridemia in
insulin-resistant animal models, but not in
normoglycemic animals (181, 182). Ciglita-
zone was taken into human trials in NIDDM
subjects, but was withdrawn because of low
potency and the appearance of cataracts in an-
imals receiving long-term exposure to the
drug. It was replaced in development with the
more potent pioglitazone (183), which has
subsequently received marketing approval in
the United States and much of the world.
2.3.2.5 Structure-Activity. The relationship
of structure to hypoglycemic activity and acti-
vation of the various PPARs with substituted
thiazolidinediones and related compounds has
been the subject of intensive investigation
(179, 180, 183-202). Some general structure-
activity relationships are apparent (Fig. 1.4)
from a comparison of the common features
of the more potent compounds identified in
these studies. Thiazolidinedione hypoglyce-
mic agents can be viewed as being composed
of an acidic head group connected to a li-
pophilic tail by a phenoxyalkyl linker. The
pK, value for thiazolidinediones is about 6.8,
and thus these compounds are partially ion-
ized a t physiological pH. This appears to be
important, given that removal of the acidic
function by N-methylation leads to loss of
activity. Other acidic moieties, heterocyclic
groups like oxazolidinediones and particu-
larly a-substituted carboxylic acids, can also
2 Current Drugs on the Market

Lipophilic tail Phenoxyalkyl linker Acidic head group

0

Figure 1.4. Common structural features found in thiazolidinedione PPARy agonists and related
compounds.

replace the thiazolidinedione ring. The a-sub- in functional transactivation assays for both
stituted carboxylic acids are often highly po- PPARa and -y (EC,, PPARa 13 nM, PPARy 4
tent, but may not be selective for PPARy. nM), whereas farglitazar (17) is highly selec-
Thus compound (16)has similar potency (195) tive (190) for PPARy (EC,, PPARa 450 nM,

PPARy 0.35 nM).Neither (16) nor (17) has
significant PPARS activity.
There is a chiral center at the 5 position of
the thiazolidinedione ring, but this is not con-
figurationally stable under physiological con-
ditions. For analogous a-substituted carbox-
ylic acids, the PPARy activity resides in the
S-enantiomer. These compounds, including
the thiazolidinediones,can be viewed as deriv-
atives of phenylpropionic acid by combining
the acidic head group with the phenyl group of
the linker. Some arylacetic acids (18 and 19)
are also fairly potent PPARy agonists (201,
202), although the SAR has not been as thor-
oughly explored. Compound (18)is highly se-
lective for PPARy, whereas (19)activates both
PPARS and - y.
A phenoxyethyl group (Fig. 1.4, n = 2) as
the central phenoxyalkyl linker is commonly
found to yield highly active compounds in SAR
studies of hypoglycemic thiazolidinediones.
Often shorter chain lengths (Fig. 1.4, n = 1)or
inclusion of the phenoxyethyl group into a het-
erocyclic ring also leads to active compounds.
In the lipophilic tail, incorporation of a wide
variety of mostly aromatic and heteroaromatic
groups has produced active agents. In a very
Insulin and Hypoglycemic Agents
limited study (1861, the hypoglycemic potency
in a series of oxazolidinediones with variations
in this lipophilic tail was found to increase
with increasing log P.
A small number of 3D-QSAR studies (203,
204) on the thiazolidinediones have been re-
ported, but these agents and the PPAR recep-
tors are not particularly well suited for this
type of analysis. 3D-QSAR studies are highly
dependent on alignment, which can be diffi-
cult with very flexible molecules that bind to a
receptor principally by large regional diffuse
hydrophobic interactions.
X-ray crystallographic studies (205-210) of
thiazolidinedione and related ligands cocrys-
tallized with various PPAR ligand-binding do-
mains (LBDs) and often a fragment of the co-
activator protein SRC-1 have provided the
basis for a detailed understanding of binding
conformation, PPAR receptor specificity, re-
ceptor shape, and the ligand-activation pro-
cess. Rosiglitazone (205) binds to the PPARy
LBD in a roughly U-shaped conformation (Fig.
1.5) in a large (-1440 A3) convoluted pocket.
The thiazolidinedione ring binds in a polar
site, making hydrogen bonds with groups in
the side chains of His-449, Tyr-473, His-323,
and Ser-289. Each of these amino acids forms
part of a different helical component of the
protein. Agonist binding results in the stabili-
zation of a conformation in which the terminal
carboxylate group of the AF-2 helix at the C-
terminus of the PPARy LBD is positioned to
form hydrogen bonds with elements of the
RXR protein in the heterodimer. In addition,
it places the LxxLL motif of the coactivator in
position to bury its Leu residues in a hydro-
phobic cleft of the LBD. NMR studies (211)
 Current Drugs on the Market
W igure 1.5. A portion of the X-ray structure (205)
of rosiglitazone co-crystallized with the PPARy li-
ga~nd-bindingdomain (LBD) and a fragment of the
COactivator protein SRC-1, showing the bound con-
fo:rmation of rosiglitazone. Coordinates were ob-
tained from the Protein Data Bank (212) and dis-
pl;ayed with RasMol.
SUlggest that agonist ligand binding results in
I
a population shift in a dynamic ensemble of
coinformations rather than a switch between
siingle active and inactive conformations.
PIPARa, -6, and - y are structurally quite simi-
la r, and the agonist-binding conformations of
th e LBD are nearly identical. The P P A R y se-
le,ctivity of the thiazolidinediones manifests
SUlbtle differences (206, 207). The binding
PCcket of PPARS is narrower in the region of
AlF-2 and unable to accommodate the thiazo-
liclinedione ring or large substituents a to the
carbonyl in carboxylic acid head groups. In
PI'ARa, substitution of a larger tyrosine for
the histamine (His-323)in P P A R y involved in
h~rdrogen bonding to the thiazolidinedione
Prevents binding.
1. a-Clucosidase Inhibitors
a-Glycosidase inhibitors (Table 1.9) delay the
di1gestion of dietary carbohydrate in the form
of starch and sucrose into monosaccharides,
wllich can be absorbed from the small intes-
tirle. By delaying absorption, these agents
101wer postprandial blood glucose and insulin
l e~~e l sand are used for this purpose in the
trt?atment of type 2 diabetes.
2.4.1 Side Effects, Adverse Effects. Gastro-
intestinal disturbances in the form of flatu-
lence, abdominal discomfort, and, to a lesser
extent, diarrhea are common side effects of
therapy with a-glucosidase inhibitors. Use of
acarbose at higher doses (100 mg or greater)
has been associated with a low incidence of
elevated serum transaminase levels, most of-
ten in patients weighing less than 60 kg.
2.4.2 Absorption, Distribution, Metabolism,
and Elimination. a-Glucosidase inhibitors ex-
ert their action in the intestinal tract, and sys-
temic pharmacokinetics do not directly indi-
cate the availability of the drugs at their site of
action. Acarbose is extensively degraded in the
intestinal tract by digestive enzymes or intes-
tinal microorganisms, and in humans, less
than 2% of an orally administered 14C-labeled
dose was absorbed as the intact drug. An av-
erage of 51% of the radioactivity was excreted
in the feces, whereas 35% was found in urine
as at least 13 metabolites. These metabolites
appear to be various 0-methylated, O-sul-
fated, and 0-glucuronidated derivatives of
4-methylpyrogallol (20) (213). Peak plasma
concentrations of radioactivitv" were found 14
to 24 h after dosing, whereas the plasma con-
centration of active drug reached a maximum
in about 1 h. On i.v. administration, 89% of the
dose is excreted in the urine as active drug
within 48 h.
Low doses (25 mg) of miglitol are com-
pletely absorbed, but absorption is saturable
and is incomplete at higher doses. Peak
plasma concentrations occur in 2-3 h. The vol-
ume of distribution, 0.18 Lkg, is consistent
with distribution primarily into extracellular
water and binding to plasma proteins is negli-
gible. Miglitol is renally excreted as un-
changed drug, with a plasma elimination half-
life of 2 h.
 Table 1.9 Structure and Pro~ertiesof a-GlucosidaseInhibitors
USP or
Nonproprietary Trade
Name Name Manufacturer Chemical Class Structure Dose Duration

Acarbose Precose Bayer 25-100 mg Postprandial

3x daily

Miglitol Glyset Pharmacia Deoxynojirimycin 25-100 mg Postprandial

W and Upjohn derivative 3x daily
N with meals

Voglibose 0.2-0.3 mg Postprandial

3 X daily
2 Current Drugs on the Market
Voglibose is very poorly absorbed. In ani-
mal studies that use radiolabeled compound, it
is excreted primarily in the feces as unchanged
drug with less than 5% of the dose excreted
renally (214).
2.4.3 Physiology and Pharmacology. The
Western human diet generally includes a daily
intake of about 400 g of carbohydrate, which is
approximately 60% starch, 30% sucrose, and
10%lactose. Before absorption, dietary carbo-
hydrates must be hydrolyzed enzymatically to
monosaccharides in the gastrointestinal tract.
The enzymes that cleave a-glycosidic linkage
between C-1of a glucose unit and C-4 or C-6 of
the adjacent glucose in starch or the fructose
C-2 in sucrose are termed a-glucosidases, and
temporary inhibition of these enzymes delays
the formation of absorbable glucose from
larger carbohydrate species. Competitive in-
hibitors of a-glucosidases are used in the
treatment of type 2 diabetes to reduce the rate
of appearance of glucose in circulation after a
carbohydrate-containingmeal and thus to re-
duce postprandial hyperglycemia. Clinically,
acarbose reduces fasting plasma glucose by
1.4-1.7 mmol/L, postprandial glucose levels
by 2.2-2.8 mmol/L, and HbA,, values by
0.7-1% (3). Miglitol appears to be rather sim-
ilar. Thus these agents, although less effica-
cious than sulfonylureas or metformin, reduce
fasting as well as postprandial hyperglycemia,
probably through an indirect mechanism.
Several different a-glucosidases are re-
quired for digestion of dietary carbohydrate.
Salivary and pancreatic a-amylases are en-
doglycosidases that hydrolyze interior a(1,4)
glycosidic linkages in the glucose chains of the
two major types of starch: (1)amylose, a linear
a(l,4)-linked glucose polymer; and (2) amylo-
pectin, a branched glucose polymer containing
a(1,6)linkages at branch points in addition to
the a(1,4). Pancreatic a-amylase does not
cleave amylopectin chains a t or near the
a(1,6)-linked branch points, and neither su-
crose nor lactose is hydrolyzed. The action of
pancreatic &-amylaseon dietary carbohydrate
thus yields a mixture composed mainly of oli-
gosaccharides containing small linear a(1,4)-
linked glucose polymers (maltose, maltotriose,
etc.), the a-limit dextrins {oligosaccharides
containing 5 to 9 glucose units terminating a t
or near a glucose attached to the chain by the
a-amylase inert a(1,6)glycosidic linkage), su-
crose, and lactose.
These oligosaccharides are hydrolyzed to
absorbable monosaccharides by" the action of a
group of enzymes on intestinal brush border
epithelial cells. Hydrolysis of sucrose and
starch-derived oligosaccharides is accom-
plished by two homologous enzyme com-
plexes, sucrase-isomaltase and maltase-glu-
coamylase, which have overlapping substrate
specificities (215). Both are exoglycosidases,
hydrolyzing the terminal glucose from the
nonreducing end of an oligosaccharide. Su-
crase-isomaltase cleaves both a(1,4) and
a(1,6) linkages in small gluco-oligosaccha-
rides, debranching the a-limit dextrins and
hydrolyzing a(l,4)-linked maltose and malto-
triose. The sucrase subunit of the complex hy-
drolyzes sucrose to fructose and glucose.
Maltase-glucoamylase also hydrolyzes mal-
tose and larger linear a(l,4)-linked glucose oli-
gosaccharides. In vivo, the maltase-glucoamy-
lase complex is believed to account for almost
all glucoamylase activity, 20% of maltase, and
1% of isomaltase activity, whereas the sucra-
se-isomaltase complex accounts for all of the
sucrase, most of the isomaltase, and about
80% of the maltase activity.- Lactose is cleaved
by a third enzyme, lactase, to glucose and
galactose.
Therapeutically useful a-glucosidase inhib-
itors delay glucose absorption by temporarily
inhibiting one or more of these digestive en-
zymes. Complete inhibition of extended dura-
tion is not desirable because this leads to
unacceptable symptoms of carbohydrate mal-
absorption (cramps, flatulence, abdominal dis-
tension, diarrhea) resulting from carbohy-
drate fermentation by colonic bacteria. For in
vitro assessment of a-glucosidase activity, in-
hibition of the hydrolytic activities of intesti-
nal brush border membrane preparations or
pancreatic homogenates toward various sub-
strates (maltose, isomaltose, sucrose, etc.) are
used in quantitative assays (216). Table 1.10
lists Ki values (24, 216) for sucrase, isomalt-
ase, &d glucoamylase inhibition by acarbose
and miglitol. Perhaps because of its oligosac-
charide-like structure, acarbose is most potent
at inhibition of glucoamylase activity, whereas
monosaccharide-like miglitol is most active

Table 1.10 a-GlucosidaseInhibition by
Acarbose and Miglitola
Acarbose Miglitol
Ki ( d f l Ki (M
Sucrase 0.086 0.99
Isomaltase 0.36 46.3
Glucoamylase 0.21 0.009
"Refs. 143,144.
against sucrase. Acarbose also inhibits pan-
creatic a-amylase (68% inhibition at 4 N),
whereas miglitol inhibits this enzyme only at
very high concentrations (216). Voglibose is a
very potent inhibitor of maltase and sucrase
activity (K, values of 3.8 and 2.0 nM, respec-
tively), but also has little effect on pancreatic
a-amylase (217). Lactase is inhibited only at
very high concentrations by miglitol, and not
inhibited by acarbose.
In addition to delaying glucose absorption
by retardation of carbohydrate digestion, the
antidiabetic activity of a-glucosidase inhibi-
tors may be partly mediated by alterations in
the release of incretins. These intestinal hor-
mones, such as glucagon-like polypeptide 1
(GLP-1) and gastric inhibitory polypeptide
(GIP), are released in response to nutrients
and have insulinotropic activity as well as
many other regulatory effects. Both acarbose
and voglibose increase circulating levels of
GLP-1, whereas a-glucosidase inhibition ap-
pears to suppress GIP (218-222).
2.4.4 Structure-Activity. The structural
requirements for a-glucosidase inhibition,
particularly in series of compounds related to
those in Table 1.9, have been extensively in-
vestigated, and much of this work has been
summarized in an excellent review (223).
Analysis is complicated, however, by a number
of factors. First, several a-glucosidases that
have differing but somewhat overlapping sub-
strate specificities and differing affinities for
various inhibitors are involved in intestinal
carbohydrate digestion. Thus, no single Ki
value describes "a-glucosidase" inhibition.
Second, the inhibition kinetics of the enzymes
are complex (224, 225). The Ki values deter-
mined for competitive inhibitors of glucoamy-
lase-maltase, for instance, were substrate
(maltose or maltooligosaccharides) depen-
Insulin and Hypoglycemic Agents
dent, contradicting the classical model of com-
petitive inhibition and suggesting the exis-
tence of subtrate-induced binding modes.
Finally, a therapeutically useful a-glucosidase
inhibitor must delay, but not block com-
pletely, digestion and absorption of dietary
carbohydrate. For this reason, favorable on-
off rates and pharmacokinetics are as impor-
tant as high affinity of the inhibitor for the
target enzymes in the drug optimization
process.
Enzymatic cleavage of the glycosidic link-
age in the substrates for a-glucosidases is be-
lieved to proceed through a pair of high energy
oxonium ion intermediates (illustrated with
maltose in Fig. 1.6, Intermediates I and 111,
which must be stabilized by interaction with
the enzyme to increase the rate of the reac-
tion. Similar interactions with protonated
amine functions of a-glucosidase inhibitors oc-
cupying positions normally filled by the oxo-
nium ions stabilize the binding of the nonhy-
drolyzable inhibitor to the enzyme (223,226).
Historically, the lead compounds in the series
that led to the marketed a-glucosidase inhibi-
tors in Fig. 1.6 were identified through
random screening, but can be seen as pseudo-
glucosylamines related to high energy inter-
mediate I (inhibitor type I) or as l-deoxynor-
jirimycin derivatives related to high energy
intermediate I1 (inhibitor type 11).
Compounds (211, (221, and (23) are moder-
ate to highly potent inhibitors of intestinal su-
(21) validamine
1C50sucrase 7.5 x
IC50 maltase 1.1x
crase and maltase (217,223) and fall into the
category of inhibitor type I pseudogluco-
sylamines. Valiolamine (231, which can be rec-
ognized as a structural component of
voglibose, is the most potent of the three, sug-
gesting that the non-glucose-like 5-hydroxyl
substituent greatly enhances affinity for su-
crase. Valienamine (22), a component of acar-
 2 Current Drugs on the Market

Pseudoglucosylarnines

H h:L
O
OH
~ ~
OH H2
Voglibose

OH

s:w >
H2
Acarbose

High energy intermediate I

HO~o O HO OH

HO~o OH
) High energy intermediate I1

Miglitol

1-Deoxynorjirimycin derivatives
Figure 1.6. Relationship of a-glucosidase inhibitors to high energy intermediates in hydrolysis of
maltose.

(23) valiolamine
(22) valienamine
sucrase 7.5 x
ICbOsucrase 5.3 x maltase 1.1 x
IC50 maltase 3.4 x 10w4

shown that both the position amine function

bose, is the least potent. SAR studies that use and the presence of glucoselike hydroxyl sub-
derivatives of these compounds in intestinal stituents are essential for good sucrase inhibi-
sucrase and maltase inhibition assays have tion.

Compounds (24) and (25) are disaccharides
incorporating the methyl glycoside of the sec-
(24) a-methylacarviosin
IC50sucrase 1.6 x lo-'
ICbOmaltase 3.2 x
(25)
IC5,sucrase 1.0x lo-'
IC50maltase 4.9 x
ond sugar unit in the acarbose tetrasaccharide
structure into the inhibitor structures. Addi-
tion of the second sugar unit yields a modest
improvement in IC,, for sucrase inhibition
with a much greater improvement in that for
maltase.
Interestingly, compounds incorporating a
glucose-like 6'-hydroxyl group were slightly
less potent than (24) and (25). In fact, the high
potency of (27) for both sucrase and maltase
inhibition suggests little contribution to in-
hibitor-binding affinity from groups other
than the 3'-hydroxyl, which, comparing (26)
and (27), must be correctly oriented, and rel-
atively simple groups lacking a ring structure
(26)
Ic5,sucrase 1.6 x
IC50maltase 1.6 x
Insulin and Hypoglycemic Agents
but incorporating this feature (e.g., voglibose,
28) are quite potent.
(27)
IC50sucrase 5.2 x lo-'
IC50maltase 6.1 x lo-'
(28) voglibose
1C5,sucrase 4.6 x lo-'
1C50maltase 1.5 x lo-'
Acarbose, in vitro, is somewhat less effec-
tive than a-methylacarviosin (24) in sucrase
inhibition (223), and thus the two additional
glucose units in acarbose compared with (24)
contribute little to the affinity of the inhibitor
for this enzyme. This is in agreement with ki-
netic studies showing two saccharide unit
binding subsites at the sucrase catalytic cen-
ter of intestinal sucrase-isomaltase (225).
Glucoamylase-maltase appears to have four
such sites (227), whereas pancreatic a-amy-
lase has five glucose unit binding subsites
(228). Acarbose derivatives with additional
glucosyl substituents are potent inhibitors of
pancreatic a-amylase, but have not shown
therapeutic utility.
Miglitol is an inhibitor type I1 derivative of
1-deoxynorjirimycin (29). The parent com-
(29) deoxynorjirimycin
pound has potent inhibitory effects in porcine
intestinal mucosal preparations on a-glucosi-
References
(30) emiglitate
dase enymes. Concentrations in a narrow
range of 9.6 X lop8to 2.2 X 10W7M inhibited
50% of the sucrase, maltase, isomaltase, and
glucoamylase activity.
Many derivatives of (29) have been prepared
and tested, particularly for inhibition of porcine
intestinal sucrase activity (223). A small alkyl
nitrogen substituent is allowed, but activity de-
creases as chain length is increased from methyl
to propyl, increasing again for very long 11- or
12-carbon alkyl chains. Introduction of a double-
bond allylic to the nitrogen increases the inhib-
itory effect. Polar subtituents, with the excep-
tion of hydroxyethyl (miglitol)and aryloxyalkyl
as in (301, generally decrease sucrase inhibitory
effect. In vivo, compound (30) (emiglitate, rat
sucrase IC,, value of 0.41 produces long-
lasting inhibition. Substitutions at the 1 or 5
-position with polar substituents like hydroxy-
methyl (compound 31, mouse sucrase IC,, value
of 0.08a, and 32) also affords strong inhibi-
tors.
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CHAPTER TWO

Peptide and Protein Hormones,

peptide Neurotransmitters, and
Therapeutic Agents
VICTOR J. HRUBY
CATHERINE GEHRIG DE CHAVEZ
MALCOLM KAVARANA
University of Arizona
Department of Chemistry
Tucson, Arizona

Contents
1 Introduction, 46
2 Background, 47
3 Methodology, 48
3.1 Peptide Synthesis, 48
3.2 Peptide Conformation, 48
3.3 Peptide Topography, 51
3.4 Topographical and Conformational
Constraints, 52
3.5 Bioassays, 52
4 Examples, 53
4.1 Parathyroid Hormone, 53
4.1.1 Structure and Function, 53
4.1.2Overall View of the Structure of PTH,
54
4.1.3 Importance of Alal and Val2 in Signal
Transdudion, 55
4.1.4 Importance of A.2' and Arg5in
Receptor/Ligand Interactions, 55
4.1.5 Importance of Gly12 in Ligand-
Receptor Actions, 55
4.1.6 Truncated PTH Analogs: Identification
of the Minimum Sequence
Requirements, 56
4.1.7 Conformationally Constrained Cyclic
Analogs of hPTH(1-31)Amide, 58
4.1.7.1 In Vitro Agonist Studies, 58
4.1.7.2 In Vivo Studies, 59
Burger's Medicinal Chemistry and Drug Discovery 4.2 Melanotropins, 59
Sixth Edition, Volume 4: Autocoids, Diagnostics, 4.2.1 Structure-Activity Relationships, 59
and Drugs from New Biology 4.2.2 Studies Leading to the Identification of
Edited by Donald J. Abraham the Minimum Active Sequence of a-
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc. MSH, 60
 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
4.2.3 Importance of ~ - P hof
e~MT-11 in
Binding and Biological Activity, 61
4.2.4 Effects of P-Substituents in Biological
Potency, 62
4.2.5 Ring-Expanded Analogs of MT-II,63
4.2.6 Novel Constrained Cyclic Ladarn
Analogs of a-MSH. Agonists, 64
4.2.7 Antagonists, 65
4.3 Oxytocin and Vasopressin, 67
4.3.1 Structure-Activity Relationships, 67
4.3.2 Oxytocin, 67
1 INTRODUCTION
It is interesting to contemplate how ignorance
and misunderstanding can sect what people
learn or do not learn in science, and how this
influences what gets done in science. No where
is this more evident than in the medicinal
chemistry, organic chemistry, and biochemis-
try of peptide and protein hormones and neu-
rotransmitters and their relationships to
chemistry, biology, and medicine. Despite the
fact that at least 50% of current drugs have, as
their therapeutic target, receptors whose nat-
ural ligands are amino acids, peptides, and
proteins, most chemists know little about pep-
tide chemistry in its many manifestations
(structural, synthetic, biochemical, biological,
etc.). As a result what they believe, as shown
in Table 2.1, is often an inaccurate reflection
of current reality. This is not to say that there
are still not many problems to solve in the
areas of peptide, protein, and peptidomimetic
drug design and development. Indeed there
are problems, and they will require state-of-
the-art scientific investigations by people
knowledgeable about what has been accom-
plished and what is the nature of the problems
in the larger context of health and disease.
However, if the current attitudes persist
and most scientists are not educated in the
peptide sciences, and do not know how to de-
sign, synthesize, and evaluate peptides and
peptidomimetics with desirable chemical and
biological properties, the current ignorance
and prejudice will become a self-fulfilling
prophecy. Considering that Emil Fisher, per-
haps the greatest organic chemist, was the fa-
ther of peptide and protein chemistry, and
that two peptide hormones, insulin and oxyto-
4.3.3 Structure-Activity Relationships of
Oxytocin, 67
4.3.4 Oxytocin Antagonists, 68
4.4 Delta Opioid Fteceptor Ligands, 69
4.4.1 Analogs of Enkephalins that Lead to
Receptor-Specific Ligands and
Nonpeptide Ligands, 70
5 Things to Come in Peptide and Peptidomimetic
Design, 75
6 Acknowledgments, 76
cin, had such a dramatic affect on twentieth
century medicine, with millions of people hav-
ing taken these two hormones, often in life-
saving or enhancing situations, and with min-
imal toxicity compared to that of most other
drugs, it is strange that so many medicinal
chemists, natural products chemists, and or-
ganic chemists have paid so little attention to
this area. Nonetheless, peptide science has the
potential to produce hundreds of new drugs
that can affect all of our major diseases, in-
cluding cancer, pain, addictive behaviors
(drugs, food, cigarettes, alcohol, sex, etc.), car-
diovascular disease, mental illness, autoim-
mune diseases, and prion diseases to name
some of the most important.
In this chapter we provide a few examples
of the use of peptide science for examining
peptide and protein hormones and neuro-
transmitters. Some of the compounds we
could have included (e.g., insulin) are dis-
cussed elsewhere in these volumes. We will
concentrate on examining four examples from
the literature that we hope will illustrate both
the potential of peptide and protein chemistry
to solve therapeutic problems, and the impor-
tant considerations in design, synthesis, con-
formational/topographic-biologicalactivity re-
lationships, and other factors that have to be
considered in peptide and peptide mimetic de-
sign. In our considerations we will concen-
trate, because of space limitations, on a few
bioactive peptide hormones and neurotrans-
mitters that have led to therapeutic agents or
that show great promise for doing so. It should
be emphasized that many important and use-
ful reviews and overviews about this subject
have been written (Refs. 1-25 are 25 such ref-
erences that have been useful to us in prepar-
2 Background

Table 2.1 Commonly Held Beliefs about Peptide Drugs

Belief
1. Peptides are unstable
2. Peptides are readily attacked by protease
3. Peptides are not bioavailable
4.Peptides are too expensive
Comments
Many are because they are biological switches or
substrates, but they can easily be made stable.
Many are for the reason in 1, but again this can easily be
remedied.
Most peptides and proteins are not orally bioavailable,
but many are >SO% bioavailable, given in the lungs,
through the eyes, transdermally, i.v., end so forth.
The cost of manufacturing peptides has decreased
dramatically in the last 20 years and can be expected
to decrease even more in the future.

ing this chapter), and should be referred to by
the interested reader. Many of these provide
important additional insights and information
regarding the design, synthesis, and confor-
mational structure-biological activity aspects
of this research. In addition a few books/mono-
graphs have appeared that provide much use-
ful information (27, 28), including the nine-
volume series The Peptides (29-371, which,
although somewhat out of date, still provides
much essential information on synthetic,
structural, and molecular pharmacological/
medicinal aspects of this area.
2 BACKGROUND
The discovery of peptide and protein hor-
mones and peptide neurotransmitters began
in the late nineteenth century with the discov-
ery of the pituitary principles (now referred to
as oxytocin, vasopressin, ACTH, a-mela-
notropin, etc.). However, at the time it was not
thought that these "principles" were peptides
or proteins. That they were was first firmly
established by duvigneaud and colleagues
with the sequence of oxytocin (38) followed
shortly thereafter by its total synthesis (39). A
few years later Sanger et al. (40) determined
the primary structure of insulin. These two
seminal discoveries ended all the controver-
sies, and established that peptides and pro-
teins could be hormones, and later, peptide
neurotransmitters were similarly discovered.
Since then hundreds of peptide and protein
hormones and neurotransmitters have been
identified and their structures determined.
After duvigneaud's proof of the biologically
active structure of oxytocin by total synthesis
and the demonstration of its equivalence to
natural oxytocin by chemical, biological, and
clinical studies, many thought the next task
was to continue synthesis of the ever more
complex peptides and proteins whose primary
structures were being determined. However,
duvigneaud had other ideas. He thought that
his structure determination and synthesis
were only a beginning, and set out immedi-
ately to modify the structure of oxytocin by
total synthesis (e.g., see Ref. 41). Thus began
what we call today de novo ligand-based drug
design. Important early contributors to this
approach were Joseph Rudinger (42) and Rob-
ert Schwyzer (2). Work from their laboratories
and many others led to the concepts of struc-
ture-activity relationships, which we take for
granted today. Over time it has become clear
that not only structural considerations (func-
tional groups; acidbases; hydrophobesthydro-
philes; methyl, ethyl, propyl, isopropyl, etc.)
were important, but that conformational con-
siderations also were critical components of
biological activity, especially with regard to
molecular recognition, transduction, stability
against proteases, and bioavailability. This
has led to the development of a strategy of
conformational constraints that continues to
be further developed today. Because many of
the constraints make use of some kind of cy-
clization, it is critical to consider the synthesis
of medium size rings and macrocyclic rings as
part of a strategy for developing peptide and
peptidomimetic drugs. Despite considerable
success, there are still many unexplored areas
involving design and synthesis of these cyclic
structures. In this chapter we outline how
48 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
these considerations have been used to design
bioactive peptides that can lead to therapeutic
agents.
3 METHODOLOGY
The successful development of peptide, pro-
tein, and peptide mimetic biologically active
hormones and neurotransmitters depends
critically on five chemical aspects:
1. Robust synthetic methods, including asym-
metric synthetic methodology.
2. Considerations of peptide conformation.
3. Considerations of side-chain conforma-
tions, particularly of pharmacophore moi-
eties (topography).
4. Critical use of conformational and topo-
graphical space.
5. Proper choice and use of biological assays.
We briefly discuss each of these with partic-
ular reference to peptide, protein, and peptide
mimetic hormone and neurotransmitter de
novo design.
3.1 Peptide Synthesis
The development of peptide synthesis meth-
odology during the past 40 years has been
spectacular, to the point where today it is per-
haps the most robust organic synthetic meth-
odology, particularly through the use of the 20
amino acids commonly used by genes to con-
struct proteins. It is now possible, using opti-
mized chemistry, to construct a peptide of 20
to 40 amino acids by stepwise synthesis with
an average yield at each coupling step of
>99%. Although one could draw attention to
many important synthetic developments to
account for this robust chemistry, perhaps
three major developments were most impor-
tant. First was the introduction of the N"-tert-
butyloxycarbonyl group for temporary protec-
tion of the a-amino group of the incoming
amino acid during assembly of a polypeptide
chain (43,44).Second was the development of
the solid-phase synthetic method by Merri-
field (45). Initially, the solid-phase method
was severely criticized by most organic chem-
ists, including most synthetic peptide chem-
ists, because "intermediates" were not puri-
fied. This stimulated Merrifield and others to
optimize every step in solid-phase synthesis to
today's levels of nearly 100% completion in
many cases (for a few excellent reviews, see
Refs. 46-49; for an excellent book, see Ref.
50). Ironically, nowadays many organic and
medicinal chemists use solid-phase organic
synthesis to make their favorite organic mole-
cules on solid supports. Seldom, however, do
they optimize their syntheses. This leads to
significant impurities that need to be re-
moved. Finally, the third major reason for the
excellent progress in peptide synthesis was the
introduction and development of high pres-
sure liquid chromatography (HPLC). The
thousands of theoretical plates on HPLC col-
umns allow rapid purification of peptides after
synthesis of the polypeptide chain and re-
moval of the side chain protecting groups. Re-
verse-phase HPLC has been particularly use-
ful for the purification of most peptides.
3.2 Peptide Conformation
A central dogma of molecular biology is that
the biological activity of a peptide or protein is
directly related to its three-dimensional struc-
ture. In the postgenomic era, this dogma will
take center stage as we seek a more critical
understanding of the chemical/physical basis
for life and the underlying chemistry related
to diseases. A clear understanding - of the rela-
tionships of peptide structure to conforma-
tions in biological systems is critical for future
progress. It is often stated that small (<lo res-
idues) linear and cyclic disulfide-containing
peptides have "random" conformations in so-
lution. This is a misunderstanding of the na-
ture of peptide and protein conformations. In
this regard, the prescient insights of Ram-
achandran and coworkers regarding the en-
ergy of peptide and protein backbone confor-
mations stands out as perhaps the greatest
insight to date into peptide and protein back-
bone conformations (51-53). By simple steric
considerations and evaluation of the energy
landscape of backbone phi (4)and psi (+) an-
gles (the bond omega angle was considered to
be a "rigid" trans conformation, except for X-
Pro where it could be cis) (Fig. 2.1), they de-
termined that a good deal of 4-+ torsional
space was of high energy and therefore not
3 Methodology 49

amino acid residue

-C N- -C N-

O o
9- trans
g-: = -600 t: x1=+18O0

and chi-1 k(-),g ( + ) ,

Figure 2.1. Definitions of the conformations of phi ($1, psi (+), omega (o),
trans] conformation.

.ccessible to most amino acid residues in a
leptide (Gly is an exception). For L-amino ac-
is, a relatively small number of low energy
onformations were available, including a-he-
k (right-handed and left-handed), P-sheets
parallel and antiparallel), extended confor-
nations, and p-turns [for definitions of differ-
nt types see Lewis et al. (54,5511. At the times
f these predictions, the high resolution X-ray
nalyses of peptide and protein structures
rere minimal. Since then, thousands of X-ray
structures of peptides and proteins have been
determined, and essentially all residues in
polypeptides (Gly excepted) are found near the
low energy conformations found on a Ram-
achandran plot (a plot of C$ and $ torsional
angles as a function of energy). From this in-
sight several tentative hypotheses can be con-
sidered in the design of peptide hormones and
neurotransmitters [and for many other pep-
tides that are either part of a larger protein or
function in other ways (e.g., as epitopes for the
50 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
Table 2.2 Kinds of Conformational Constraints Leading to Specific Secondary Structures
for Peptide Hormones and Neurotransmitters
Structural Constraint
N-terminal to C-terminal cyclics
Cyclic disulfides
Cyclic ladams and lactones: side-chain to side-
chain, terminal to side-chain, and so forth
Cyclic backbone: side-chain; cyclic
backbone-backbone, backbone to C- or
N-terminal
C"-alkyl or aryl groups
Bulky amino acid side-chains (e.g., naphthyl,
adamantyl, etc.)
immune system, as substrates for proteases,
etc.)]:
1. Because the targets for hormones and neu-
rotransmitters are integral membrane pro-
teins such as G-protein-coupled receptors
(GPCRs), the hormones will bind to these
proteins with specific secondary struc-
tures.
2. The solution conformation will adapt to the
conformation that leads to greatest (lowest
energy) interactions with the receptor.
3. When bound to the receptorlacceptor, the
peptide will be an integral part of the recep-
tor conformation (i.e., behave as a "mini"
protein).
4. Agonists, competitive antagonists, and
competitive inverse agonists bind in the
binding pocket of the receptor differently
(have different structure-activity relation-
ships and different pharmacophores),
which leads to different structures for the
receptor-ligand complex in each case.
A way to test these hypotheses directly
would be to examine by X-ray crystallography
or high resolution nuclear magnetic resonance
(NMR) spectroscopy, the three-dimensional
structure of the peptide-protein complexes.
However, examination of the high resolution
structure of integral membrane proteins has
turned out to be very difficult, not only be-
cause of their reasonably large size (40-80
Ex~ectedOutcome/Comments
Cyclic hexapeptides and pentapeptides p-turns,
y-turns.
Cyclic 11-, 14-, 17-, 20-, and 23-membered rings:
stabilize p-turns, y-turns, p-turnslp-sheets.
Stabilize &-helices,p-turns.
Stabilize p-turns, y-turns, p-sheets.
Stabilize a-helix, 3,,-helix, extended structure
depending on R groups.
Limit 4, JI angles; stabilize transannular interactions;
define pharmacophore space.
kDa), but even more so because they primarily
are biologically viable in the anisotropic envi-
ronment of the membrane bilayer. Chemists
and physicists have yet to develop robust
methods to examine three-dimensional struc-
tures at high resolution (<2.5 A) in these en-
vironments. Hence, ligand-based drug design
in this area has been largely excluded. None-
theless, a highly successful alternative ap-
proach has been developed for peptides, which
can be referred to as peptide ligand-based drug
design with conformational constraints. The
kinds of conformational constraints that have
proved to be most successful are shown in Ta-
ble 2.2. Examination of this table indicates
that constraints using cyclizations of various
kinds often require the use of novel amino ac-
ids, C"-substituted amino acids, and/or the use
of novel bulky side-chain amino acids for suc-
cess. Importantly, proper choice of conforma-
tional constra&s can lead to small peptide
analogs whose three-dimensional structure in
solution can be determined by state-of-the-art
two- and three-dimensional NMR methods,
with insight from circular dichroism spectros-
copy, FT-infrared spectroscopy, and Raman
spectroscopy. X-ray crystallography also has
provided important conformational informa-
tion. In addition, modern computational
chemistry methods with appropriately modi-
fied force fields can provide predictive confor-
mational insights that are helpful in the de-
sign of biologically active ligands.
3 Methodology
L-tyrosine
Figure 2.2. Plot of energy map of versus 2 for tyrosine and of (2S,3R)P-methyl-2',6'-dimethyl-
tyrosine, where each contour represents 1 kcal from the lowest energy conformation.
3.3 Peptide Topography
In the case of most peptide hormones and neu-
rotransmitters, the key pharmacophore ele-
ments are often the side-chain groups that
make up the peptide structure. In the vast ma-
jority of cases aromatic residues of Phe, Tyr,
Trp, and His; amino residues from the N-ter-
minal; the €-aminogroup or Lys and the gua-
nidyl group from Arg; and/or the carboxyl
groups from Asp, Glu, or the C-terminal car-
boxylate play key roles in the pharmacophore.
This poses unique problems that have begun
to be investigated only recently. The problem
is that the energy landscape for the side chains
of amino acid is much flatter than that in
phi-psi space. As shown in Fig. 2.1 each chi
angle starting with X , has three possible side-
chain conformations: gauche(-), trans, and
gauche(+); and, as shown in Fig. 2.2, although
indeed the g(- ), trans, and g(+)conforma-
tions are the low energy conformations, the
energy difference between them is small, and
the energy barriers between them are small
and easily accessible at 300°C. Thus it is very
difficult to predict which side-chain conforma-
tion the receptorlacceptor prefers, or whether
it makes any difference in terms of molecular
recognition in binding affinity, transduction,
or biological activity in general. To investigate
this problem Hruby et al. initiated a compre-
hensive program of topographical design con-
straint (18) of side-chain groups in amino ac-
ids. To accomplish this goal they and others
developed methods for the asymmetric syn-
thesis of P-substituted amino acids (e.g., p-Me,
-Et, -i-Pr-Aryl,etc.) and in the case of Phe and
Tyr also 2'- and 6'-methyl substituted amino
acids (18, 56-60). Because there is a second
chiral center created for most of these novel
amino acids, we and others have had to de-
velop novel synthetic methodology to prepare
the desired designed amino acids (see Fig. 2.3
for some examples). Constraints in chi space
and 2) of various degrees are found in the
derivatives of phenylalanine (1,2, 3, and 4;
Fig. 2.3), tyrosine (1, 2, 3, and 4; Fig. 2.3).
Because many of the novel amino acids have
chiral centers at the a- and p-carbons, they
exist as four isomers: (S,S),(5'31, (R,S), and
( R p ) . We have shown by both NMR (e.g., see
Ref. 61) and computational chemistry (18)
that significant constraints are introduced be-
tween the g(-), trans, and g ( + ) conforma-
tions, and that there are significant energy dif-
ferences between them in many cases (18).We
provide specific examples of the use of such
topographically constrained amino acids in de
novo peptide design in Section 4 of this chap-
ter. Suffice it to say at this point, that such
constraints can provide unique insights into
 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
Figure 2.3. R2= -H, -cH~,
-Alkyl, -Aryl
novel chi-constrained amino acids (80-88)
for topographical design (related
lead references). (4)
the topographical requirements of pharma-
cophore side-chain groups (for reviews, see
Refs. 18, 56).
3.4 Topographical and Conformational
Constraints
From the above discussion it is clear that op-
timal design of biologically active peptides re-
quires the use of both conformational and to-
pographical constraints in the same molecule
to obtain insights about the physiologically ac-
tive conformation. Because only a limited
number of topographically constrained amino
acids are commercially available, studies that
make use of both types of constraint simulta-
neously have been rather limited. However, in
Section 4 we illustrate the power of this meth-
odology to provide peptide and peptidomi-
metic analogs with unique biological activi-
ties. As work proceeds on the proteome and
with the observation that molecular recogni-
tion is critical in virtually all life processes, we
believe that the use of conformational and to-
R = -CH3, -Aryl n = 0,1,2,3,4 etc.
(89-91) R = Me, Aryl, etc.
pographical considerations will be an integral
part of any drug or ligand design project. It
seems to be particularly important for the de
novo design of a nonpeptide ligand based on a
peptide pharmacophore (e.g., see Refs. 18, 92,
93).
3.5 Bioassays
Central to any program in ligand-based drug
design or in the development of novel confor-
mationally and/or topographically con-
strained biologically activity peptides is the
need for robust bioassays. Currently, high
throughput screening is touted as the best
method for discovering drug leads. Often this
involves only binding affinity studies, and
sometimes a second-messenger assay as well.
Although this is often a good starting point to
get a micromolar binding lead compound, it
has become increasingly clear that for treat-
ment of diseases, especially those involving
peptide hormones and peptide neurotransmit-
ters, the adaptive changes that occur in the
4 Examples 53

Table 2.3 Biochemical and Biological Assay for Peptide Hormones and Neurotransmitters
- --

Assay Considerations
Binding affinity For potency and selectivity must use multiple receptor types and
subtypes, membranes or cells or pure receptor.
Second-messenger assays CAMP,GTP-yS, and so forth: membranes, cells, or tissues;
agonist vs. antagonist vs. inverse agonist.
Bioassays Tissues: provide functional information (e.g., smooth muscle;
heart; liver; brain; etc.).
In vivo assays Assess functional or behavioral effects (e.g., blood pressure;
diuresis; pain; heart rate; respiration; pigmentation).
Behavioral assays Assess changing behaviors (e.g., pain; addiction; feeding; sexual;
aggression; learning; etc.).

endocrine system or in the central nervous
system render the relationships of hormone or
neurotransmitter bioactivity different in nor-
mal and diseased states. As a result more ex-
tensive assays that address the adaptive
changes that have occurred as a result of dis-
ease are needed (e.g., see a discussion of
changes that occur in pathological pain states
in Ref. 94). Thus more extensive and perhaps
different assays will be needed. In Table 2.3 we
outline some of the assays that should be used
and some considerations related to these as-
says.
4 EXAMPLES
4.1 Parathyroid Hormone
4.1.1 Structure and Function. The parathy-
roid hormone (PTH) is a linear polypeptide of
84 amino acids (Fig. 2.4) and plays a critical
physiological role in bone growth, renal func-
tions, and calcium homeostasis (95-98). This
peptide, along with the active forms of vitamin
D, constitutes the principal regulator of cal-
cium homeostasis in humans. PTH increases
the concentration of extracellular (blood) cal-
cium through a concerted effect primarily on
bone, intestine, and kidneys. Lowering of
blood calcium levels stimulates production of
PTH, which consequently works to maintain
appropriate levels of blood calcium through
three distinct mechanisms: (1)increased rate
of dissolution of the bone mineral; (2)reduced
clearance of calcium by the kidneys, by return-
ing more of the calcium filtered by the glomer-
ulus back to blood; and (3) increasing the effi-
ciency of calcium absorption in the intestine
through an indirect mechanism involving the
activation of vitamin D (98). In addition, PTH
plays a role in maintaining the concentrations
of inorganic phosphates in the body.
PTH is biochemically synthesized as a pro-
hormone, which differs from PTH by the fact
that it has six additional residues at the ami-
no-terminus. The exact function of the pro-
hormone sequence is unclear but it has been
hypothesized that the production of PTH as a
prohormone is important for the efficient
packaging of the hormone. Although calcium
ion concentrations in blood appear to play a
significant role in the regulation of PTH, other
agents such as magnesium, epinephrine, and
dopamine also play a role in regulating PTH
secretions in vivo.
Recently, a related peptide, the parathyroid
hormone-related protein (PTHrP) containing
140 amino acids was identified as a secretory
product of tumors associated with the clinical
syndrome of humoral hypercalcemia of malig-
nancy. This peptide hormone is also produced
by a variety of normal cells and acts as a para-
crine andlor autocrine regulator in develop-
ment (99, 100). PTHrP appears to play a key
role in early skeletal development in the em-
bryo, in cell differentiation (99),and also facil-
itates the transport of calcium across the pla-
centa and during lactation. Both PTH and
PTHrP have high affinity for the parathyroid
hormone receptor (PTHlR), a G-protein-cou-
pled, seven-transmembrane receptor (GPCR)
with nearly equivalent potency. The fact that
PTH and PTHrP, despite sharing only limited
sequence homology, binds to and activates the
same receptor (PTHlR) suggests that both
54 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agen

Activation domain Inhibitory domain

15

ASN ASP GLU LYS LYS ARG PRO ARG GLN SER

SER HIS GLN LYS SER LEU GLY GLU ALA ASP LYS ALA

LN PRO LYS ALA LYS ILE LEU

Figure 2.4. Schematic representation of the functional domains of parathyroid hormone molecule.

peptide hormones assume very similar bioac-
tiie conformations when bound to the recep-
tor.
The importance of both peptide hormones
in controlling various physiological processes,
and the observation that their malfunction
can result in disease states such as hypercal-
cemia, osteoporosis, cancer, renal and cardiac
problems, as well as problems associated with
Hkeletd development has stimulated research
in the design and development of stable and
potent synthetic analogs of these hormones as
therapeutics. Hence, extensive structure-ac-
tivity relationship (SAR) studies aimed at elu-
cidating amino acid residues in PTH or
PTHrP that are important for binding and po-
tency have been undertaken. The SAR studies
have shown that the N-terminal portion, con-
sisting of the first 34 amino acids, possesses
full activity in both PTH and PTHrP (100a).
This review focuses on SAR studies that
have helped in understanding the molecular
basis for ligand-receptor interactions and il-
lustrates key linear, truncated, or cyclic an
logs that agonize or antagonize the PTH r
ceptor. Both classes of compounds have be€
used to study the mechanism of action of tl.
hormone in normal and pathophysiologic
states. Some specific potential therapeutic a
plications of these compounds might incluc
treatments for acute hypercalcemia arisir
from parathyroid adenoma, hyperplasia, ar
carcinoma. Highly selective and potent anta
onists of PTH also have been proposed i
therapeutic agents for the treatment of, fc
example, chronic hyperparathyroidism, oste
porosis, and fibrous dysplasia.
4.1.2 Overall View of the Structure of PTI
Parathyroid hormone is an 84 amino acid, li:
ear polypeptide secreted by the parathyro
gland. Early structure activity studies show(
that the amino-terminal region consisting
34 residues was sufficient for full biologic
activity (96, 97). This fragment could furthl
be divided into three distinct regions: (1)TI
4 Examples
N-terminal region which is helical in nature
and plays a key function in hormone stimu-
lated adenylate cyclase activity in vitro and
receptor activation in vivo; (2) The middle
'hinge' region which is possibly involved in a
beta-turn and is responsible for the proper ori-
entation of the truncated peptide in the recep-
tor pocket; and (3) A carboxyl terminal do-
main which is also helical and contains
residues important for binding and receptor
recognition (101b, 102).
4.1.3 lmportance of Ala' and ValZ in Signal
Transduction. SAR studies have implicated
the C-terminus domain to be i m-~ o r t a n for
t these results illustrate the importance of the
maintaining potent receptor-binding affinity.
The more flexible N-terminus region appears
to play a critical role in signal transduction.
Deletion of the first two amino-terminal resi-
dues results in a loss of all, or virtually all,
biological activity but not in a loss of binding
affinity to the PTHl receptor in vitro. Thus
there appears to be an apparent structural
separation of binding and activation regions in
PTH.Deletion of Alal was accompanied by a
marked decrease in adenylate cyclase activity,
implying that the minimum sequence re-
quired for biological activity starts at the sec- peptides undergo reverse-turn structures (55,
ond amino acid.
ZO z5
4.1.4 lmportance of Arg and Arg in Re-
ceptorhigand interactions. Early SAR studies
had indicated that conserved structural mod-
ifications in the central portion of the active
fragment of PTH-(1-34) were remarkable for
their relative lack of effect on biological activ- followed by a p-turn at positions 12-15 in PTH
ity. To evaluate the biological role of Ar2'
and ArS5in PTH-(1-34), fragment-selective
postsynthetic modifications of these two resi-
dues in hPTH-(1-34) active fragment were
done by use of 1,2-cyclohexanedione (100).
1,2-Cyclohexanedionereacts specifically, com-
pletely, and reversibly with the guanidino
group of arginine, to give a single product,
N7,N8-(1,2-dihydroxycyclohex-1,2-ylene)argi- Aib12 gave agonists with binding affinities
nine, [DHCH-AI-~~,DHCH-A~$~]~PTH-(~-
34) (103). This modified analog showed, at
most, 16% of the activity of the unmodified
hormone hPTH-(1-34), based on in vitro renal
adenylate cyclase assays using borate buffer.
Reversal of arginine modification completely
restores biopotency, indicating that the po-
tency decline observed after 1,2-cyclohex-
anedione treatment is not caused by nonspe-
cific alterations of structure. The near total
loss in biopotency of the modified hormone
may be further explained by the increase in
steric bulk of the Arg20,25side chains or by a
change in the formal charge of the guanido
group from a + 1to zero in a putative arginine-
borate complex. Whether these two effects in-
dividually or in combination may cause a dra-
matic decrease in biopotency through adverse
effects on hormone-receptor interactions or
through the induction of a conformational
change in the peptide is not clear. However,
positively charged guanidino group in Arg on
binding and bioactivity.
4.1.5 lmportance of ClylZ in Ligand-Recep-
tor Actions. Glycine, the simplest amino acid,
plays a very important role in proteins and
peptides. The absence of a side-chain func-
tional group renders Gly a high degree of con-
formational flexibility in peptides. In addition,
this amino acid can also access the phi and psi
space of D-amino acids. Hence, glycine is gen-
erally found in the region where proteins and
104,105). Glycine is the 12th residue from the
N-terminus in the PTH sequence (106, 107).
This residue is highly conserved in PTH and
PTHrP isolated from all species to date. Struc-
tural studies that use the Chou-Fasman anal-
ysis and both CD and NMR studies have pre-
dicted that the a-helical N-terminal domain is
and 9-12 in PTHrP. Thus, the biological con-
sequences of single-residue changes at posi-
tion 12 were assessed in vitro, to determine
the role of this residue in hormone-receptor
interactions and its contribution toward the
bioactive conformation of the peptide. In the
human hormone [T~r~~]hpTH-(l-34)-NH,
substitution of Gly12 by Ala12, ~-Alal,and
similar to that of [Tyr34]hPTH-(1-34)-NH2in
bovine renal cells (Kb [T~r~~]hPTH-(1-34)-
NH, = 0.7 d ; Kb for substituted analogs be-
tween 0.7 and 1.0 d). The Pro12analog binds
approximately 840-fold less tightly than
[Tyr3*]hPTH-(1-34)-NH,. The K, values for
adenylate cyclase activity are also similar to
56 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agenb

Table 2.4 Binding and Cyclase Activity of Selected Antagonist Analogs of PTH
with Bone-Derived ROS1712.8 Cells
Analog Binding,K, (nM) Cyclase K, (nM) Ref.

that of [Tyr34]hPTH-(1-34)-NH2 for the introduction of hydrophobic side-chain groups

Ala12,~-Alal,,and Aib12analogs, whereas the
K, value for the Pro1' analog is 3500-fold
lower (107).
To design potent and selective antagonists
(108) of PTH or any other peptide ligand, it is
necessary to identify the sites within the hor-
mone where structural modifications can be
made that maintain high receptor affinity, but
without inducing agonist properties. In prin-
ciple these goals can be achieved by two differ-
ent routes: (1) stabilize the antagonist in a
conformation favored by the receptor for mo-
lecular recognition but not transduction; and
(2)introduce new structural moieties that will
add binding elements that interact with the
receptor at sites different from or in addition
to those present in the native agonist. These
binding elements should retain the "bioactive
conformation" of the ligand required for re-
ceptor binding that does not lead to informa-
tion transduction.
Previous SAR studies with human and bo-
vine PTH have shown that removal of the first
six amino-terminal residues gives an analog
with potent antagonistic activity in vivo, such
as [ T ~ ~ ~ l h P T H - ( 7 -NH,
3 4 ) (109) (Table 2.4). within the native peptide sequence that are
Substitution of Gly12in this fragment by Ala12
and ~-Alal,gives analogs that are about twice
as potent as the parent peptide [ T ~ r ~ ~ l h p T Hand- critical region necessary for full biological
(7-34) NH, in both binding and CAMP assays,
whereas the Pro1, analog is approximately
twofold less active vs. the parent peptide. Fur-
thermore, replacement of Gly12 with D-Trp
(Table 2.4) results in an antagonist that is ap-
proximately 10-fold more potent in the bind-
ing assay and 12-fold more potent at inhibiting
agonist-induced (3 nM [Nle8*18,Tyr341bPTH- tested for their ability to competitively inhibit
(1-34)NH2) CAMP activity (Table 2.4). In con-
trast, the L-Trp analog (110) failed to alter ei-
ther binding or cAMP activity, suggesting that
in the D-configuration at position 12 provides
auxiliary hydrophobic interactions with the
receptor, which promotes binding. A combina-
tion of bulky hydrophobic residues at position
12 together with the substitution of Met8 and
Met1' by Nle gave analogs having binding ac-
tivity 10- to 15-fold more potent than that of
[Nle8,18,Tyr341bPTH-(7-34)NH2. These re-
sults suggest that introducing amino acid res-
idues with aliphatic and/or bulky hydrophobic
side chains in either the D- or conformer can
improve antagonism in the 7-34 truncated se-
ries (Table 2.4).
4.1.6 Truncated PTH Analogs: Identification
of the Minimum Sequence Requirements.
Truncation of peptide hormones having more
than 15 amino acid residues to smaller yet bi-
ologically active and potent analogs is very of-
ten a challenging task. Most often truncation
not only allows researchers the flexibility of
synthesizing large numbers of analogs with
considerable ease, speed, and at less cost, but
the truncated analogs also can be studied
more systematically to elucidate the regions
important for binding and bioactivity. SAR
studies of PTH have shown that the minimum
and binding activity resides in the first 34
amino acids (1-34) of the polypeptide (109,
110). To further determine the residues
within this polypeptide fragment that play an
important role in molecular recognition, sev-
eral truncated synthetic fragments of bovine
parathyroid hormone (bPTH) were made and
the binding of 1251-labeled[Nle',Nle18,Tyr34]
bPTH-(1-34)amide, as well as serve as antag-
onists of PTH-stimulated cAMP in vitro (111).
4 Examples
Table 2.5 in Vitro Biological Potencies of Truncated Analogs of Bovine
Parathyroid Hormone
Peptide
[Nle8,Nle18,Tyr34]bPTH-(334)amide
[Nle8,Nle18,Tyr341bPTH-(7-34)amide
[Nle8,Nle18,Tyr341bPTH-(10-34)amide
[Nle8,Nle18,Tyr341bPTH-(15-34)amide
[Nle8,Nle18,Tyr341bPTH-(2034)amide
[Nle8,Nle18,Tyr34]bPTH-(25-34)amide
[Nle8,Nle18,Tyr341bPTH-(2&34)amide
"The concentration of each peptide that inhibited specific binding of radioligand to a half maximal level was taken as the
apparent binding constant (Kb) of that peptide.
bMolarratio of inhibitory peptide to PTH required for 50% inhibition of CAMPin vitro (112).n.d., not determined.
'Testing at a concentration of peptide as high as that used in the binding assay was precluded. Nonspecific in bition of
CAMPactivity at peptide concentrations greater than 4 x
Table 2.5 summarizes the results of this study.
The inhibition of specific binding of radioli-
gand was assessed for each peptide fragment
over a concentration range from 1 X 10-lo to 1
x 10-'M.
The binding studies indicate that deleting
the first two residues from the amino-terminal
completely eliminates any detectable biologi-
cal activity, while maintaining potent affinity
for the receptor (112). Further stepwise
amino-terminal deletions from position 3 to 27
caused a progressive decrease in binding avid-
ity. However, it was observed that all of the
truncated analogs, except the [Nle8,Nle18,
Tyr34]bPTH-(28-34) amide fragment, could
completely and specifically inhibit the binding
of radioligand when added at sufficiently high
concentrations (1 x lop2 M). This suggests
that a small chain of 10 residues (25-34), but
not as small as a seven amino acid fragment, is
able to satisfy all the requirements for binding
to the receptor pocket (113). The 25-34 se-
quence was found to be conserved in hor-
mones isolated from all species studied to date,
indicating that this fragment contains impor-
tant structural information for binding. The
observation that the relative order and magni-
tude of the apparent binding constants ob-
served correlates closely with the Ki values for
each peptide fragment in the in vitro adenyl-
ate cyclase assay suggests that the mechanism
of hormone antagonism caused by these pep-
tide fragments is through direct competition
between PTH and analog fragment for recep-
tor occupancy. Competition for receptor-bind-
ing sites by the analog fragments is not attrib-
K, (Renal),M" Ki (CAMP),M [Analogll[PTHlb
1.2 X 1 . 2 lo-@
~ n.d.
1.1X lo-6 3.0~ 30
2.7 X 7.0~10-~ 70
3.2 x lo-' 3.7~10-' 370
7.3 x lo-6 7 . 0 lo-5
~ 700
3.4 x lo-4 No inhibitionc >4000
No binding Not tested n.d.
M.
uted to nonspecific peptide effects, given that
hPTH-(44-68) and hPTH-(53-84) fragments
do not inhibit binding of radioligand at a max-
imal concentration of 1 x M.
A second major direction toward the devel-
opment of potent and biologically active ago-
nists of PTH relies on the incorporation of D-
amino acids in place of the naturally occurring
L-enantiomer at selected positions along the
native peptide sequence. This technique has
been successfully used to generate stable,
more potent and bioavailable analogs of vari-
ous peptide hormones and other biologically
active peptides (e.g., Refs. 114-117). Four an-
alogs of PTH having D-amino acids at selected
positions along the native peptide sequence
were synthesized and tested for their ability
hi to
bind and activate the PTH receptor. Because
earlier studies (118-120) had shown that even
subtle changes near the amino- and carboxyl-
termini conferred large changes in biopotency,
the D-isomers were introduced in these re-
gions. Table 2.6 summarizes the results of key
analogs. substitutions at the carboxyl-termi-
nus are very well tolerated, given that replace-
ment of Tyr34 by its D-isomer gave a peptide
that is biologically equipotent. In addition,
this peptide may be more active in vivo than in
vitro because of its greater resistance to enzy-
matic degradation. Structural modifications
in the amino-terminal activation region are
poorly tolerated in terms of biopotency. Sub-
stituting Val2 by its D-isomer in [ ~ - T y r ~ ~ ]
bPTH-(1-34) gave analog [ ~ - V a l ~ , ~ - T y r ~
bPTH-(1-34) amide, which lacked nearly all of
the in vitro biological activity compared to
58 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents

Table 2.6 Biological Activity of Bovine PTH(1-34) and bPTH(2-34) in Rat Renal
Adenylate Cyclase Assay
Substitution Fragment Potency" Relative Potency (%Ib
None 1-34 5400 (3,90043,000) 100
[Tyr341 1-34 amide 16000 (11,000-23,000) 300
[~-Tyr~~l 1-34 amide 14500 (11,000-17,000) 270
[D-T~~~~I 2-34 amide 130 (120-150) 3
[D-V~',D-~~~] 2-34 amide 90 (60-100) 2
[~-Val~,~-Tyr~~] 1-34 amide 80 (60-100) 1
"Potency estimates are based on three independent assays except for [D-Tyrm1bPTH-(1-34)amide. Limits in parentheses
represent SEM for each peptide except [~-Tyr~~]bPTH-(134)amide, for which 95% confidence limits are provided.
bRelative potency calculated on the basis of the mean potency with the activity of the reference peptide, unsubstituted
bPTH-(1-34) taken as 100%.

that of the unsubstituted analog. A similar ef- cells in culture. Cyclization was achieved
fect is seen when the first amino-terminus res- through the formation of side chain to side
idue is deleted, as in [~-Tyr~~]bPTH-(2-34) chain lactam bridges at three distinct sites
amide. However, no additivity of effects (in within the 17-29 residue sequence of hPTH-
terms of a further reduction in biopotency) (1-3l)amide, previously identified by high res-
was seen on combining ~-Val' and [Tyr341 olution NMR to be involved in ion-pairing in-
bPTH-(2-34)-amide in the same peptide frag- teractions in solution (121). There are two
ment (Table 2.6). distinct ion pairs in the C-terminal a-helical
region that could potentially contribute to-
4.1.7 Conformationally Constrained Cyclic ward the stability of the helix. These are be-
Analogs of hPTH(1-31 )Amide tween Glu22-Lys26and LysZ6-Asp3' and have
4.1.7.1 In Vitro Agonist Studies. Cycliza- (i, i + 4) spacing, whereas the third ion pair
tion at specific sites within a peptide is often between Lysz7and Asp3' having (i, i + 3) spac-
used as tool for enhancing the selectivity, ing is helix destabilizing. Lactam bridges be-
binding, and biopotency of the peptide ligand tween these residues would restrict their con-
(3-5, 8-11). Cyclization introduces con- formational flexibility, thus reducing the
straints that can stabilize the bioactive confor- number of possible conformations the peptide
mation of the ligand, thus improving the bio- can assume in solution.
logical properties, and can also help to identify Table 2.7 lists the results of in vitro CAMP-
regions within the peptide sequence that may stimulation studies in ROS cells for the cyclic
be important for molecular recognition (3-5). lactams as well as for the linear [Leuz7]hPTH-
Given that the biologically relevant three-di- (1-3l)amide analog. Cyclic peptide (51,having
mensional structure of PTH is at best specu- (i, i + 3) spacing, shows half the in vitro po-
lative, cyclic lactam analogs of hPTH, de- tency of its linear counterpart l. In (5)a twist
signed to stabilize the amphipathic helical is introduced into the helix on lactam forma-
region between residues 17 and 30, and known tion and may be responsible for destabilizing
to be important in binding to the receptor the helix and thus lowering the potency of this
(120), were synthesized and tested for their analog (Fig. 2.5). The increased bioactivity of
ability to activate rat osteosarcoma 1712 (ROS) analog 2 versus 1 is not attributed to an in-

Table 2.7 Biopotencies of Cyclic Constrained Analogs of hPTH(1-31) Amide in ROS Cells
Peptide Adenylate Cyclase Activity, EC,, (nM)
4 Examples
(a) MI8
A: Peptide 3; B: Peptide 4; C: Peptide 5.
Figure 2.5. Models showing ladam positions in receptor-binding region of human PTH.
crease in the a-helical content of (2) (based on
CD data), but rather is attributed to the re-
placement of a polar lysine residue (LysZ7)on
the hydrophobic face of the helix by a hydro-
z
phobic leucine residue (Leu 7). Constraining
analog (2) through a lactam bridge between
G1uZzand Lys2" (analog 3) further improves
bioactivity, presumably through stabilization
of the a-helix. Although peptides (3) and (4)
both have lactam bridges between the (i, i + 4)
residues, and thus have the same helical con-
tent, the CD study indicates differences in the
:onformation populations. Peptide (3)presum-
ably has a more nearly perfect helix than that of
~eptide(4), probably because of the fact that in
:4)the constraint is toward the terminus to the
lelix region. This difference may account for the
oss in bioactivity of (4) versus (3).
4.1 J.2 In Vivo Studies. The bioavailabili-
.ies of the cyclic analogs were tested by
neasuring their abilities to lower the blood
Iressure in rats when injected either intrave-
lously or subcutaneously at a dose of 0.8
lmo1/100 g of the analog (122). In addition, the
,ime required by these analogs to lower the
dood pressure to a maximum extent was also
neasured as a factor to evaluate their in vivo
!fficacies. Intravenous injections of the cyclic
~nalogselicited relative hypotensive effects
lot significantly different from those observed
from the CAMP study. In addition, the time
required to attain minimum blood pressure
was not significantly different for these
analogs.
However, subcutaneous injections pro-
duced quite different results. By this proce-
dure, peptides (I),(2),and (5)produced signif-
icantly lower drops in blood pressure than did
hPTH-(1-34)-NH, and peptides (3) and (4).
However, the times required for attaining
maximal drop in blood pressure after subcuta-
neous injections are quite different, in that an-
alog (3)reached the minimum blood pressure
much faster than either (4) or (5), a result
consistent with data from CAMPstudies. Fi-
nally, it was noticed that analogs truncated to
the first 30 amino-terminal residues had poor
biological properties such as hypotensive ef-
fects in rats when administered subcutane-
ously. However, these peptides showed similar
hypotensive properties when administered in-
travenously, suggesting that Val3' might be
playing an important role in the transport of
these analogs from the site of injection into the
vascular system.
4.2 Melanotropins
4.2.1 Structure-Activity Relationships. The
melanotropins, a-, p-, and y-melanocyte stim-
60 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
ulating hormone (MSH) are a group of endog-
enous neuropeptides that control various key
physiological functions through their interac-
tion with the five transmembrane G-protein-
coupled receptors (GPCRs),called the melano-
cortin receptors (MCRs). To date, five such
receptors (123-129) have been identified: the
MC1-R a-MSH receptor (pigmentation recep-
tor); the MC2-R [ACTH receptor; recent re-
search has revealed that the MC2-R binds
ACTH with high affinity, but that the MC2-R
does not bind MSH peptides (130)l; and the
MC3-R, the MC4-R, and the MC5-R, whose
functions are under intensive investigation.
a-Melanotropin (a-MSH), Ac-Ser-Tyr-Ser-
Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-
NH,, was one of the first peptide hormones
isolated from the pituitary gland (131-135).
This hormone plays an important role in skin
pigmentation. Other effects ascribed to this
hormone include: (1)regulation of the release
of pituitary and peripheral hormones, such as
somatostatin and corticotropin; (2) sebotro-
phic effects, adrenal steroidogenesis, immune
response, cardiovascular and metabolic ef-
fects; and (3)important roles in the CNS such
as (a)control over learning, memory, motiva-
tion, sleep, sexuality, and food intake (associ-
ated with obesity); (b)effects on neurotrans-
mission such as cholinergic and dopaminergic
systems; (c) neurochemical effects; (d) hypo-
thermic and antipyretic effects; (e) effects on
nerve regeneration; and ( f ) interactions with
other neuroactive compounds such as opiates.
It is now well established that the MC1 re-
ceptor mainly expressed in melanocytes and
leukocytes plays a key role in skin pigmenta-
tion and inflammatory response (136, 137).
The MC2 receptor is expressed only in the ad-
renal gland and mediates glucocorticoneogen-
esis (123). The MC3 and MC4 receptors are
both found in the brain, the MC3 receptor is
found in the arcuate nucleus and the nucleus
of the solitary tract, whereas the MC4 receptor
is mainly found in the hypothalamus (138).
Finally, the MC5 receptor is involved in exo-
crine functions and is localized predominantly
in the sebaceous glands (139). The complex
role of the melanotropins and the MC recep-
tors, in controlling various physiological func- generation cyclic analogs replaced the Met4
tions, has made it difficult to draw simple cor- and Gly1° residues with a pseudoisosteric Cys
relations between these receptors and the
biological effects of their ligands. Hence, re-
search has been focused on developing potent
and receptor-specific ligands that have well-
established biological properties. Thus, potent
and selective agonist and/or antagonists could
serve as valuable tools for determining un-
equivocally the roles of the melanocortin re-
ceptors in humans.
4.2.2 Studies Leading to the Identification
of the Minimum Active Sequence of cv-MSH.
Early SAR studies with a-MSH were aimed at
elucidating the residues in the tridecapeptide
that were most responsible for its biological
activity. Although previous reviews (140) have
covered the results of these studies, it is
worthwhile to point out some of the salient
features.
1. Replacing Met4 by norleucine (Nle4) in-
creases the potency of the resulting mela-
notropin, probably because of the chemi-
cally inert and hydrophobic side chain of
Nle. Early studies had shown that Met is
easily oxidized to its sulfone, which in-
creased the hydrophilicity dramatically
and thus decreased the bioactivity of the
corresponding derivative in the amphibian
skin assays.
2. The substitution of Phe 7 by ~ - P h ein-~
creased the biopotency of the resulting an-
alog, suggesting that a reverse p-turn con-
formation (1411, formed in the active core
sequence (His-Phe-Arg-Trp) of a-MSH,
may be important in binding and activity.
Consequently, Hruby et al. developed the
first highly potent and stable analog of
a-MSH, [Nle4,~-Phe7]-a-MSH [NDP-MSH,
MT-I, (Ac-Ser-Tyr-Ser-Nle4-Glu-His-~
Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2)l (141).
This compound was found to be a potent
agonist of the MC1 receptor in amphibian
and lizard skin assays.
To further improve the biological efficacy of
a-MSH analogs, and to test the p-turn hypoth-
esis, a series of cyclic compounds with greater
conformational rigidity compared to that of
the linear analog were synthesized. The first-
+ Cys residues, to give c[Cys4,Cys10]-a-MSH
4 Examples
(142). This compound was found to be more
potent than a-MSH, but lacked prolonged bi-
ological potency. In view of the importance of
melanotropic peptides in treatment of pig-
mentory disorders and as a tool to detect mel-
anoma cancer, quenched dynamic simulation
studies (143) were undertaken to develop
a-MSH analogs with superpotent and pro-
longed biological activity in the lizard skin and
tyrosinase assay. The key observations (143)
from these studies were the following: (1)both
a-MSH and NDP-a-MSH assumed folded con-
formations with a probable P-turn-like struc-
ture at residues seven and eight; (2)the hydro-
6
phobic
g
side-chains of His , D- or ~ - P h eand~,
Trp occupied one face of the folded molecule
with the charged (hydrophilic) side-chains of
Glu5, Arg, and Lysl1 segregated toward the
opposite face of the molecule; (3) although
5
Glu and Lysl1 occupied the same face, their
oppositely charged side-chains were not in
proximity for strong coulombic interaction.
The molecular mechanic studies also indi-
cated that if Lysl1 was moved to replace Glylo,
the side-chain of Lys1° could be involved in a
strong electrostatic interaction with the side-
5
chain of Glu , and such an interaction may
further stabilize the folded conformation and
thus improve binding. These observations led
to the development of A ~ [ N l e ~ , ~ - P h e ~ , L ~this ~ l - (His6, Phe7, Aqf, Trp ) play an
a-MSH and A~[Nle~,Asp~,~-Phe~,L~s~~]-a-
MSH analogs, which both assumed folded con-
5
formations with Asp (Glu5) and Lys10 side-
chains in proximity to each other when tested
Figure 2.6. Structure of the potent nonselec-
tive human melanocortin receptor agonist MT-
11.
by molecular dynamic calculations (143, 144).
Analog A C [ N ~ ~ ~ , A ~ ~ ~ , D - P ~ is ~ ~ ,
approximately fivefold more potent than
a-MSH in the lizard skin assay (144). To fur-
ther improve potency the cyclic analog with a
lactam bridge connecting Asp5 and Lys1° side-
chains was synthesized and tested in frog skin,
lizard skin, and tyrosinase assays (145, 146).
Interestingly, the cyclic analog Ac-Nle4-
~[Asp~,~-Phe~,Lys~~]-NH,-(4-10)a-MSH (MT-
11, Fig. 2.6) was a superpotent agonist in both
lizard skin and tyrosinase assays, but had ap-
proximately half the activity in the frog skin as-
say (Table 2.8). MT-11 also showed prolonged
biological activity in the in vitro assay.
While these studies were in progress, a se-
ries of extensive studies on the minimum ac-
tive sequences necessary for biological activity
in melanotropin peptide at pigment cell recep-
tor. Through use of a series of assays and ani-
mals it was determined that the His-Phe-Arg-
Trp segment of a-MSH was the minimum
sequence for full agonist activity (147, 148).
4.2.3 Importance of D-Phe7 of MT-II in
Binding and Biological Activity. The bioactive
conformation of a-MSH involves a p-turn in
the message sequence and it has been pro-
posed that the side-chain residues ofg groups in
s ~ region
important role in binding receptor selectivity
and agonist activity (144,146,149,150). Thus,
it was proposed early that replacing the Phe7
residue by its D-isomer could alter the confor-
62 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents

Table 2.8 Relative Biological Potencies of a-MSH and Related Analogsa

Bioassay System
Melanotropin Analog Frog Skin Lizard Skin Tyrosinase

"All potencies reported are relative to a-MSH = 1.0 in assay systems.

bn.d.,not determined.

mation of the peptide and thereby its bioactiv-
ity, and thus earlier studies led to substitu-
tions in this position (e.g., see Refs. 151-153).
To further test this hypothesis, analogs of
MT-I1having a halogen at thepara position in
the phenyl ring and the ~ - N a l ( 2 ' analogs
)~
were synthesized and tested for activity at
mouse and human melanocortin receptors
(frog skin, mMC1-R, hMC1-R, hMC3-R,
hMC4-R, and mMC5-R; Tables 2.9 and 2.10)
(154). The para-chloro and n-para-fluoro-
phenyl analogs were agonists at both mouse
melanocortin receptors (mMC1 and mMC5)
and also showed full agonist activity in the
frog skin assay. In the frog skin bioassay
(FSB), [~-Phe(pF)~]-MT-11 (EC,, = 0.10 nM)
is 20 times more potent than [ ~ - P h e ( ~ C l ) ~ lter
MT-I1 (EC,, = 2.0 nM). However, at the
mMC1-R and hMC1-R, [ D - P ~ ~ ( ~ F ) ~ ] - M T for - I I their binding potencies and receptor selec-
has approximately one-third the potency of
that of [~-Phe(pcl)~l-MT-II at both receptors
(Table 2.9). At the hMC3, hMC4, and hMC5
receptors a similar trend was observed, with
the [~-Phe(pcl)~l-MT-II being a more potent
agonist than [~-Phe(pF)~]-MT-I1(154).
However, for the D-p-iodophenyl and the D-
Nal(2'I7 analogs the results in the FSB were
different. It was found that both of these com-
pounds were potent antagonists in the FSB.
Surprisingly, both analogs were found to be
potent agonists at the mouse MC1 and hMCl
receptors. At the hMC3, hMC4, and hMC5 re-
ceptors the [~-Phe(pI)~l-MT-11 and [~-Nal(2')~1-
MT-I1 (SHU9119) were both potent antago-
nists, with pA, = 9.3 at the hMC4R and a pA,
= 8.3 at the hMC3 receptor. On the other
hand, the [~-Phe(pI)~]-MT-11 analog showed
partial agonist activity at the hMC5 receptor,
whereas SHU9119 is a full agonist at this
mammalian receptor. It is interesting to note
that the [~-Nal(l')~l-MT-I1 analog showed full
agonist activity at all human receptors, indi-
cating that subtle differences in the topogra-
phy of the peptide ligand can significantly al-
- bioactivity. Other MT-I1 derivatives
having larger lactam rings have been tested
tivity. However, the larger, mare flexible ring
improved neither the potencies nor the selec-
tivities (at hMCRs) of these analogs (155),sug-
gesting that ligand-receptor interactions favor
a 23-membered ring.
4.2.4 Effects of p-Substituents in Biological
Potency. P-Methyl-substituted amino acids
recently have been used to study the topo-

Table 2.9 Activities of MT-I1 Derivatives at Various MC1-Rsa

EC,, Values (nM)
Compound Frog Skin mMC1-R hMC1-R
a-MSH 0.10 1.3
[~-Phe(pF)~]-MT-11~ 0.10 0.026
[~-Phe(pcl)~l-MT-II 2.0 0.0095
[~-phe(pI)~]-MT-I1 Antagonist pA2= 10.3 0.19
[D-N~(~')~]-MT-II Antagonist pA2= 10.5 0.039
"Data adapted from Ref. 154.
bMT-II:Ac-Nle-CLASP-His-D-Phe-Arg-Trp-Lysl-NH,.
4 Examples 63

Table 2.10 Activity of MT-11Analogs at Various Human Melanocortin Receptorsa

EC,, Values (nM)
Compound hMC3-R hMC4-R hMC5-R
a-MSH 669 210 807
[~-Phe(pF)~]-MT-11 191 19 1360
[~-Phe(pCl)~l-MT-I1 63 18 117
[~-phe(pI)~]-MT-I1 1134 573 684
pA2= 8.3 pA, = 9.7
[D-N~(~')~]-MT-II 2813 Antagonist 434
pA2= 8.3 pA, = 9.3
"Data adapted from Ref. 154.

graphical requirements for ligand-receptor in- three stereoisomers in the FSB. In the lizard
teractions. Hruby et al. (156) studied the skin assay [(2S,3R)-P-Me-Trpg]-MT-I1 analog
change in bioactivity that occurs on incorpo- is 33 times less potent than MT-11, whereas
rating all four isomers of p-methyl Phe and the other three isomers showed nearly equal
p-methyl Trp in the MT-I1 template (Table potency. These results demonstrate the sensi-
2.11) (154). p-Methyl analogs constrain the x tivity of the lizard skin melanocortin receptor
space and thus restrict the rotational freedom for proper stereochemistry at the p-carbon
of the side-chain group. As a result, the pep- atom and ultimate topographical presentation
tide will have certain restricted conforma- of the Phe7 residue, which contrasts with the
tions, which in turn can increase receptor- requirements at the frog skin receptor. Fur-
binding preferences. Incorporation of all four thermore, the results in Table 2.11 suggest
7
isomers of P-methyl Phe in MT-I1 generally that incorporation of constrained amino acids
led to decreased potency in the FSB by 1 to 2 into MT-I1 or other melanotropins can fur-
orders of magnitude (Table 2.11). However, in ther improve bioactivities, when these new an-
the lizard skin assay [(2S,3S)-p-Me-Phe7l- alogs possess the right side-chain conforma-
MT-I1was approximately 17 times less potent tion for binding to their target receptors.
than MT-11; the [(2S,3R)-p-Me-Phe7]-MT-I1
and the [(2R,3S)-p-Me-Phe7]-MT-I1 were 4.2.5 Ring-Expanded Analogs of MT-11. To
equipotent; and the [(2R,3R)-p-Me-Phe7l- study the "bioactive" backbone conformations
MT-I1 is 2 orders of magnitude less potent required for tight binding and receptor selec-
than MT-I1 (Table 2.11). In the p-Me-Trpgse- tivity at the human melanocortin receptors, a
ries the [(2R,3S)-p-Me-Trpg]-MT-I1 showed series of ring-expanded MT-11 analogs having
much higher potency than that of the other alanine at position 10 were synthesized (157).

Table 2.11 Comparative Biological Activities of Topographically Modified MT-I1 Analogsa

Frog Skin Lizard Skin
-

Analog EC50 (nM) Rel. Potency ECbO (nM) Rel. Potency

MT-I1 0.1 1.0 0.2 1.0
[(2S, 3s)-p-Me-Phe7]-MT-I1 6.25 0.016 3.44 0.06
[(2S, 3R)-p-Me-Phe7]-MT-I1 6.25 0.016 0.2 1.0
[ ( Z , 3s)-p-Me-Phe7]-MT-I1 2.0 0.05 0.3 0.67
[ ( Z , 3R)-p-Me-Phe7]-MT-I1 16.67 0.006 20.0 0.01
[(2S,3S)-p-Me-Trpg]-MT-I1 0.44 0.23 1.0 0.2
[(2S,3R)-p-Me-Trp9]-MT-I1 28.60 0.004 6.67 0.03
[ ( Z , 3s)-p-Me-Trpg]-MT-I1 0.06 1.6 1.43 0.14
[ ( Z , 3R)-p-Me-Trpg]-MT-I1 0.33 0.3 1.0 0.2
-

"Data adapted from Refs. 155 and 156.

bMT-II:Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lysl-NH,.
64 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agent
Table 2.12 Binding Affinities of the Ring-Expanded MT-I1 Analogsa
Analog Structure
"Adapted from Ref. 157.
The introduction of Ala1° increases the cyclic
lactam from a 23-membered (MT-11) to a 26-
membered ring, thus allowing greater confor-
mational flexibility, thought to be important
for maintaining active peptide conformation.
Furthermore, this 26-membered analog was
modified through inversion of chirality at po-
sitions 5, 7, and 9 to test the stereochemical
requirements of the side-chain groups at these
positions in receptor selectivity. These studies
showed that at the hMC1, hMC4, and hMC5
receptors, MT-I1 and [AlalO,Lysll]-MT-II(an-
alog 1) had similar binding affinities, whereas
a t the hMC3R, [Alal0]-MT-I1had a fourfold
decrease in binding potency (Table 2.12). Re-
moval of Nle4 from [AlalO]-MT-I1(analog 2)
resulted in a 5.5-fold decrease in binding affin-
ity at the hMClR and a four- and twofold de-
crease at the hMC3 and hMC4 receptors, re-
spectively. However, this analog was unable to
competitively displace [lZ5I]-NDP-MSHfrom
the hMC5R at ligand concentrations > 1000
nM. When ~ - P hine analog
~ 1 was substituted
by its L-isomer (analog 3),the binding affinity
at all melanocortin receptors was decreased
substantially. However, it was interesting to
note that this substitution resulted in a 100-
fold ligand selectivity between the hMCl
andhMC3 receptors and a 30-fold selectivity
between the hMC3 and hMC4 g
receptors. The
introduction of a D-Trp residue in place of
Trps in analog1gave a peptide with poor bind-
ing affinity at all receptors (analog 41, and the
maximum effect was seen at the hMC4R
where an 80-fold decrease in binding was ob-
IC,, (nM)
hMClR hMC3R hMC4R hMC5I
served for analog (4) versus analog (1).Fi
nally, the importance of the chirality of tht
side-chain of Asp5 analog (1)was determinec
by substituting the D-isomer (analog 5). Thir
compound was unable to competitively dis
place [lz5I1-NDP-MSH at the hMC3 anc
hMC5 receptors, and was, respectively, 750
and 100-fold less tightly bound to the hMCl
and hMC4 receptors versus analog (1).Al
peptides were agonists at the human melano.
cortin receptors.
4.2.6 Novel Constrained Cyclic Lactam An,
alogs of a-MSH.Agonists. The important roh
of a-MSH in the control of various key physi,
ological functions has prompted research ir
the development of novel tight-binding and re.
ceptor-selective linear or cyclic analogs. How,
ever, to date, many of these compounds thal
incorporate the core sequence show similar se.
lectivity profiles. The recent observations thal
the hMC3 and hMC4 receptors might be in.
volved in the control of feeding behavior and
energy homeostasis in humans has height.
ened the interest to develop selective and po.
tent agonists and/or antagonists for these re.
ceptors. The novel cyclic peptide [(O)C-CH,
CHz-C(0)-c-[His6-~-Phe7-Arg8-Trp9-Lys1
NH, (MK-1, Fig. 2.7) (158, 159) is a highly
selective and potent agonist of the hMC4 re-
-
ceptor. This compound is a modified cyclic lac-
tam analog of MT-11, the nonselective but po.
tent agonist of the melanocortin receptors. In
MK-1 cyclization is through the use of a linker
-
arm involving a backbone to side-chain cy-
 4 Examples
clization strategy. In MT-11,however, cycliza-
tion is through a lactam bridge between the
carboxyl side-chain of Asp5 at the N-terminus
and the €-amino group of Lysl0 at the C-
terminus. Binding studies showed that MK-1
is 700-fold more selective at the hMC4R vs.
hMClR (IC,, hMClR = 4200 nM and IC,,
hMC4R = 6.0 nM). Additionally, this analog is
55- and 120-fold more selective at the hMC4R
vs. the hMC3R and hMC5R7respectively (Ta-
ble 2.13). In a related study Bednarek et al.
(160) used a similar cyclization strategy to
identify a selective and potent peptide agonist
of the hMC4 receptor. They synthesized a se-
ries of cyclic peptides having the general struc-
ture cE-His-D-Phe-Arg-Trp-YI- NH,, where X
= succinyl or o-amino-carboxylic acid (as
linker arms) and Y = a-, o-diamino-carboxylic
acids. In uitro analysis identified two analogs
that showed good agonist activity and a high
degree of selectivity for the hMC4 receptor.
The first analog involved the substitution of a
2,4-di-aminobutyric acid group in place of
Lys1° and the use of a succinyl linker to cyclize
the peptide (21-membered ring). This analog
is a potent hMC4R agonist with 55-fold selec-
tivity for the hMC4R versus the hMC3R recep-
tor and is about 1000-fold more selective for
the hMC4 versus the hMC5 receptor. Another
analog having a Glu residue in place of Lys1°
Figure 2.7. A novel selective cyclic lactam
agonist of the human melanocortin 4 recep-
tor.
and using a 3-aminopropionic acid moiety as
the linker arm was also a potent hMC4R ago-
nist (EC,, = 0. 56 ? 0.04 nM) (BBRC). This
compound showed a 90-fold greater affinity
for the hMC4 versus the hMC3 receptor and
was 2000-fold more selective for the hMC4
versus the hMC5 receptor. To test the impor-
tance of lactarn ring size in selectivity and bio-
activity analogs having smaller and larger
rings were tested. The smaller lactam rings
showed lower potency, whereas it was ob-
served that rings having more than 21 atoms
showed good potency and selectivity at the
hMC4 receptor.
4.2.7 Antagonists. Although SAR studies
of a-MSH have resulted in the development of
potent agonist dnalogs in the past two de-
cades, the search for modestly potent and se-
lective competitive antagonists has not been
as successful (161-164). Although a few early
studies suggested weak or partial inhibition
by a-MSH analogs or a-MSH-related frag-
ment derivatives (132, 161, 163, 164), these
results were difficult to improve on. Since
then, the design and synthesis of a-MSH an-
tagonists has been slow because of a lack of
understanding of antagonist structure-activ-
ity relationships. Recently, SAR studies in the
Hruby group, involving the use of novel cyclic
Table 2.13 Biological Activities of the Cyclic a-MSH Analogs at Various Human Melanocortin Receptorsa
hMC3R hMC4R hMC5R
Peptideb IC,, (nM) EC,, (nM) % Activity IC,, (nM) EC5,0 % Activity Ic50(nM) EC,, (nM) % Activity
VJH-085 332.0 70 95 5.96 1.55 100 710 61.52 78
MK-2 21.46 81.9 87.8 102.8 281.6 35 154.9 148.3 100
MK-3 6.67 3.75 36 50.68 >10 pM 0.0 6118 117 100
Antagonist
MK-4 6.1 >10 pM 0.0 231 1220 22.8 27.5 1 81
Antagonist
MT-I1 1.52 1.85 100 6.86 2.87 100 7.47 2.45 100
"Adapted from Ref. 159.
bVJH-085:(0)C-(CH2)2-C(O)-c[His-~-Phe-Arg-Trp-Lysl-NH2;
-2: (O)C-(CH2)2-C(O)~[His-~-(2')Nal-Arg-Trp-Lys]-NH2;
ME-3: (0)C-(CGH,)-C(0)-c[His-D-(2')Nal-Arg-Trp-
Lysl-NH2;ME-4: (OK-(CH2),-C(O)-c[His-~-(2')Nal-Arg-Trp-Lysl-NH~.
 4 Examples
ladam analogs having side chain to backbone
cyclization, have provided new insights into
antagonist structure-activity relationships. It
was found that the substituting hydrophobic
residues at position seven of the message se-
quence (MK-2) resulted in small losses in
binding potency at the hMC4R, accompanied
by an increase in antagonist activity at this
receptor (Table 2.9). More recently it was
found that when a hydrophobic group such as
o-phthalic acid was used as the cyclizing linker
(MK-3),this analog was a potent antagonist of
the hMC4 receptor (PA, = 111, even though
MK-3 has a binding affinity of 13.5%, com-
pared to that of the superagonist MT-11, used
as a control in this study. Interestingly, both
peptide MK-2 and MK-3 had strong binding
affinity for the hMC3 receptor. Thus it ap-
pears that the combination of a bulky residue
at position 7 and a hydrophobic phenyl ring in
the linker arm (MK-1 to MK-3) significantly
alters the conformational representation of
the ligand presented to the hMC4 receptor, so
as to convert the hMC4R-selective and potent
agonist to a hMC4R-selective and potent an-
tagonist (159). These SAR studies also showed
that increasing the 23-membered lactam ring
of MK-1 by one carbon atom (succinyl vs. glu-
taric linker) gives a highly selective and potent
antagonist (MK-4) for the hMC3 receptor. At
the hMC4 receptor, analog MK-4 is only a par-
tial agonist, while concomitantly maintaining
full agonist activity at the hMC5 receptor. An-
alogs MK-1, MK-3, and MK-4 therefore rep-
resent the first examples of a class of cyclic
melanotropin ligands with high selectivity and
defined biological activities at the physiologi-
cally important hMC3 and hMC4 receptors
(159).
4.3 Oxytocin and Vasopressin
4.3.1 Structure-Activity Relationships. Oxy-
tocin and vasopressin are neurohypophyseal
hormones synthesized in the hypothalamus
and then transported along with their carrier
proteins to the posterior lobe of the pituitary
Oxytocin (OT) ~-~~s-~yr-~le-~ln-Asn-~~s-~ro-~eu-~
Vasopressin (VP) H - ~ ~ s - ~ y r - ~ h e - ~ l n - A s n - ~ ~ s - ~ r o - ~ r ~ - ~
gland. They were first discovered in the poste-
rior pituitary gland, but they also are located
in the CNS and numerous other organs. These
peptides both consist of nine amino acids; they
both contain a 20-membered disulfide and an
acyclic tripeptide tail; and the two peptides
differ only in positions 3 and 8.
Oxytocin causes contractions of uterine
smooth muscle and is secreted during labor.
Oxytocin also stimulates contraction of
smooth muscle in mammary glands, to cause
milk ejection in nursing mothers. A large per-
centage of deliveries in the United States are
induced or augmented by i.v. oxytocin (165).
Oxytocin also has some intrinsic antidiuretic
activity. Less is known about the role of oxy-
tocin in the central nervous system. It has
been shown to be involved in memory and
learning as well as grooming and sexual be-
havior. At low doses vasopressin controls the
resorption of water by the distal tubules of the
kidneys and regulates the osmotic content of
blood. At high doses it causes contractions of
blood vessels causing localized increases in
blood pressure. It also has CNS activities.
4.3.2 Oxytocin. Oxytocin was the first
peptide hormone to be used clinically. Ex-
tracts from the posterior pituitary were intro-
duced in obstetric practice more than 90 years
ago (166). Oxytocin also was the fmt peptide
hormone to be sequenced (38)and synthesized
(39). Oxytocin is, in itself, quite an effective
therapeutic agent when used appropriately,
and there has been relatively little interest in
designing more potent oxytocin agonists.
However, when administered in high doses in
a glucose solution, oxytocin can cause water
intoxication. Therefore, analogs of oxytocin
having reduced intidiuretic activity would
have some benefit.
4.3.3 Structure-Activity Relationships of
Oxytocin. Hundreds of analogs of oxytocin
have been synthesized since 1953. There have
been several comprehensive reviews of this
work (167-169).
68 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
Table 2.14 Biological Activities of Oxytocin (OT) and Selected Agonist Analogs
Rat Uterus (IUImg)
Oxytocin
Oxytocinoic Acid
Deamino-OT
[I-carbalOT
Deamino-[l-carbalOT
Deamino-[6-carbalOT
[~-Tyr'10T
[Thr410T
[Thr4, deamino-1-carba10T
[Thr4,deamino-6-carbalOT
Fragment studies of oxytocin indicated
that the 20-membered ring structure is neces-
sary for full agonist activity at the uterus re-
ceptor. The acyclic tripeptide tail, although
having no biological activity itself, is impor-
tant for binding to the uterus receptor. The
C-terminal amide group is important for activ-
ity. Replacement of the C-terminal CONH,
with COOH to form oxytocinoic acid results in
a 400-fold decrease in activity (170) (Table
2.14).
Deletion of the free alpha amino group
from the Cys in position 1 to form l-deamino-
oxytocin enhanced both the oxytotic and anti-
diuretic activities (171). Replacement of the
sulfur in Cysl with a methylene group to form
[1-carba]OTresulted in an increase in potency
(172) (Table 2.14).
The orientation of the aromatic ring of Tyr2
is important for binding and transduction.
Substituting a D-Tyr in this position results in
a partial agonist (173,174). The Tyr hydroxyl
group is also important for activity because
[Phe210Thas only approximately 1/15 the po-
tency of oxytocin (175).
Manning et al. (176) synthesized [4-threo-
nineloxytocin, which was the first oxytocin
analog containing only naturally occurring
amino acids that was more potent than oxyto-
cin itself and had lessened antidiuretic activity
(177). This indicates that an amino acid side
chain that can form a hydrogen bond at posi-
tion 4 is important for agonist activity. The
substitution of Gly in position 7 resulted in an
analog that retained its oxytotic activity, yet
had very little antidiuretic activity (178).
Combining these two substitutions to form
Reference(s)
546 181
1.3 170
803 182
734 172
1898 183
929 184
Partial agonist 173,185
900 186
272 180
695 180
[Thr4,Gly7]oxytocin resulted in an analog
with high specificity (179).
Lebl et al. (180) combined the Thr4 and
the carba substitutions, synthesizing [Thr4,
deamino-1-carbaloxytocinand [Thr4,deamino-
6-carba]oxytocin. Although these analogs had
slightly reduced potency at the uterus receptor,
they were more selective, having much lower va-
sopressor activity.
4.3.4 Oxytocin Antagonists. Oxytocin an-
tagonists would be of great therapeutic benefit
in delaying labor. Some of the earliest oxytocin
analogs demonstrated antagonistic properties
(see Refs. 167, 168, 187 for reviews). Methyl-
ation of the tyrosine hydroxyl group to produce
CMeO-Tyr210xytocinresulted in an analog with
antagonism of the vasopressor response but
not the oxytocic response (188) (Table 2.15).
Schulz and Du Vigneaud (189,190)found that
substitution of penicillamine in position 1 of
oxytocin and 1-deamino-oxytocin resulted in
potent antagonists of the oxytotic response in
uitro and partial agonistJantagonist properties
in viuo.Substitution of leucine in position 2 to
form [Pen1,Leu2]oxytocin increased antago-
nist activity (191). [D-Pen1,0rn810xytocinis
about 10-fold more potent as an antagonist
than either [D-Penl]oxytocinor [Penlloxytocin
(192). Substitution of threonine in position 4
of a series of [l-(p-mercapto-p,p-dialkylpropi-
onic acid)loxytocin doubled potency (193).
Sawyer and Manning (194) concluded that
the C-terminal glycinamide is not necessary
for oxytotic antagonists. Amino acids with
bulky side chains can be substituted for the
4 Examples 69

Table 2.15 Oxytocin (OT)Analogs with Antagonistic Potencies at the Rat Uterus In Vitro
Estimated pA2"
Values In Vitro
Analogs (NOM2+) Reference(s)
[Penl]OT 6.86 189,190
[Pen1,Leu210T 7.14 191
[~-Penl]oT 6.94 209
7.14 193
[~-Pen',orn~]OT 7.89 193
[~-Pen',Thr~]oT 7.52 179
[l-(P-Mercapto-P,P-diethylpropionic acid)]OT 7.55 179
acid),Thr4]0T
[l-(/3-Mercapto-j3,/3-diethylpropionic 7.72 179
[l-(P-Mercapto-P,P-cyclopentamethylenepropionic acid)]OT 7.61 179
[I-(P-Mercapto-P,P-cyclopentamethylenepropionic acid),Thr4]0T 7.91 179
[D-Tic210T 6.50 201
[T~r(Me)~loT 6.79 202
[~-Phe(4-Et)~lOT 8.15 203,204
[2-Br-Tyr210T 7.05 205
[3-I-Tyr210T 7.05 206
[Deamino-Per~l,Tyr(Me)~]OT 7.76 193
[Deamino-Pen1,Tyr(Me)2,0rn81vasotocinb 7.70 192
[l-(p-Mercapto-P,P-diethylpropionic
acid),Tyr(Me)2,0rn81vasotocin 8.91 207
acid),
[l-(p-Mercapto-P,P-cyclopentamethylenepropionic
~(Me)2,0rn81vasotocin 8.52 207
acid),
[I-(/3-Mercapto-P,P-cyclopentamethylenepropionic
~-Tyr(Me)~,Vd~,Cit~]vasopressin 8.61 194
acid),
des(Gly(NH2)[1-(~-mercapto-~,~-cyclopentamethylenepropionic
D-Phe2]arginine-vasopressin 7.64 194
d-~arba~[n-Phe(Me)~]OT 8.73 203
des(Gly(NH2)d-carba6[~-Phe(Me)210T 8.80 203
[Penl,n-Phe2,Thr4,0rnwT 7.23 208
[Penl,n-Phe2,Thr4,Thr5,0rn810T 7.16 208
Penl,~-Phe2,Thr4,Leu5,0rns10T 6.67 208

-
[Penlp-Phe2,Thr4,Asp5,0m8]OT 7.21 208
[Penlp-Phe2,Thr4,Tyr5,0rn8]OT 6.76 208
[Mpal,Glu4,Cys6,Lys810T(bicyclic) 8.2 195,196

"pA, is the negative algorithm of the molar concentration of antagonist that reduces the response to 2 x units of oxytocin
to equal the response to l x unit in the absence of antagonist.
bVasotocinis [S-ArglOT.

Gly in position 9 without a loss of antagonistic
efficacy (177). They also found several antiva-
sopressin analogs that were also antioxytocics.
Hill et al. synthesized a bicyclic analog of
the weak monocyclic agonist c[Mpa1,Cys6l-
c[Glu4,Lys810xytocin, which was found to
have potent antagonist activity (195-197) as
did many of its derivatives. Extensive NMR
and computational studies of this led to the
determination of the bioactive conformation
of oxytocin antagonists (198, 199) and to the
design of topographically constrained antago-
nist analogs (200) with unique biological prop-
erties and unique insight into the topographi-
cal requirements of OT receptors.
4.4 Delta Opioid Receptor Ligands
All of the current opioid drugs used for the
treatment of pain are primarily ligands for the
p-opioid receptor. Numerous studies since the
discovery of enkephalin 27 years ago (210)
have suggested that an opioid ligand that pri-
marily interacted with the 8-opioid receptor
would have far fewer of the toxicities generally
associated with the p-opioid ligand (respira-
tory depression, constipation, addiction, etc.).
70 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
Early efforts to convert enkephalin into a se-
lective 6-opioid ligand were successful in the
development of C[D-Pen2,0-Pen5]enkephdin
(211) (DPDPE) and its analogs, which eventu-
ally led to analogs that were essentially spe-
cific for 8-opioid receptors as agonists, such as
(2S,3R)P-methyl-2',6'-dimethyltyrosine-C[D-
Pen2,u-Pen5]enkephalin (212, 213). The ear-
lier aspects of this work have been thoroughly
reviewed (214) and a selective update has re-
cently appeared (215). A different kind of lead
to 8opioid ligands came from the discovery of
the deltorphins (216, 217) such as H-Tyr-D-
Ala-Phe-Glu-Val-Val-Gly- NH, that are found
in amphibian skins and have intrinsically
highly delta opioid receptor selectivity.
A quite comprehensive review of the struc-
ture-activity relationships of deltorphins has
been published (218). SAR studies of modified
deltorphin structures led to another class of
linear delta opioid receptor ligands such as
H-Tyr-Tic-Phe-Phe-OH (TIPP) (219) and
H-Tyr-Tic$[CH,NH]Phe-Phe-OH (TIPP[$])
(220) and related analogs, which also have
been recently reviewed (221). Our purpose in
this section is not to repeat or summarize
these reviews but rather to point to some as-
pects related to peptide design in conforma-
tional space for 6-receptors. Thus, we will be
highly selective in our choice of ligands to dis-
cuss structure-activity relationships and the
conformation and topographical properties
that lead to delta agonist and antagonist activ-
ity. In this regard it has recently been reported
that use of a new spectroscopic method, cou-
pled plasmon waveguide resonance spectros-
copy (CPWR) (222), allows one for the first
time to examine changes in G-protein-cou-
pled receptors protein structure parallel and
perpendicular to the membrane normal to
that accompanying binding of ligands. It was
shown for the human 6-opioid receptor that
agonist and antagonist binding leads to differ-
ent structures for the 6-opioid receptor (2101,
and that inverse agonist binding leads to yet
another conformation (223). These studies
provide unequivocal evidence that agonists,
antagonists, and inverse agonists lead to dif-
ferent conformations of G-protein-coupled re-
ceptors and suggest that the availability of
. conformational states are of critical
multide
importance to the function of G-protein-cou-
pled receptors. The precise conformational
difference remains to be determined.
4.4.1 Analogs of Enkephalins that Lead to
Receptor-Specific Ligands and Nonpeptide Li-
gands. The conversion of enkephalin to the
cyclic enkephalin analog c - [ ~ - P e n ~ , ~ - P e n ~
enkephalin (DPDPE), to give a potent and
high bopioid receptor ligand (2111, demon-
strated clearly the power of conformational
constraint for both enhancement of potency
and receptor selectivity (224). Subsequent
NMR (225) and X-ray crystallographic studies
(226,227) provided insights into the importance
of a turn conformation to 8-opioid receptor se-
lectivity and differences in conformational re-
quirements for agonists and antagonists, but
left unanswered the side-chain conformation
of Tyrl and Phe4 for potent and selective 6-opi-
oid receptor bioactivity. To examine these re-
quirements, we turned to topographical con-
straints, that is, to constraints in x1 andlor X,
space that can be made within the context of
the same backbone conformation for agonist
(and antagonist) biological activity (228). All
four P-methylphenylalanine-4 analogs (229)
and P-methyl-2',6'-dimethyltyrosine-1 (TMT)
(213) analogs of DPDPE were synthesized and
evaluated for binding affinities and biological
activities for their conformational and topo-
graphical properties. As seen in Table 2.16
for the [TMT1lDPDPE analogs, only the
[(2S,3R)-TMT1lDPDPE analog was both
highly potent and highly selective for the delta
opioid receptor. Conformational analysis that
used NMR and computation chemistry dem-
onstrated that for Tyrl, the trans ,yl conforma-
tion, and for Phe4, the gauche (-1 conforma-
tion, were critical for biological agonist
activity and potency.
In related studies, Mosberg et al. (230-232)
carefully evaluated the cyclic truncated del-
torphin analog H-Tyr-c-[D-Cys-Phe-D-Pen]-
OH (JOM-13) in a series of structure-activity
and conformational studies that used the
P-MePhe3 constraint (230) and alternative
constraints for the Tyrl position (see ref. 231
for an excellent review). These studies led to
the conclusion of the gauche (-1 side-chain
conformation for X, in Phe3 and the trans X,
side-chain conformation for the Tyrl x1 (232).
As expected the backbone conformations of
 Tald e 2.16 Binding Affinities for Delta Opioid Receptor Selective Ligands
-
IC,, or Ki (nM)
npound 6 p16 ratio Ref.

"A potent inverse agonist at the 6 opioid receptor (252).

the! DPDPE ring (14-membered) and the
Jo:M-13 ring (11-membered) were found to be
sonnewhat different. Mosberg et al. carried
these studies forward by examining the bind-
!
ingo f JOM-13 to a model of the 8-opioid recep-
I
i tor they developed (233, 234). By use of these
dels they developed a model of JOM-13
md to the &receptor (Fig. 2.8), which nicely
explains the structure-activity relationship
they had found for JOM-13.
Hruby et al. used their bioactive conforma-
tion model for [(2S,3R)TMT1]DPDPE for a
different purpose (Fig. 2.91, that is, to design
nonpeptide peptide mimetics. A major interest
of medicinal chemists is the development of
peptide mimetics (235). The concept of pep-

8%-ILE

Figure 2.8. JOM-13 (blue) in the 6-opioid receptor binding pocket (stereoview). See color insert.
[Taken from Fig. 2.9 in H. I. Mosberg, Biopolymers (Peptide Science), 51, 426 (1999). Reprinted by
permission of John Wiley & Sons.]
 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents

Aromatic Pharmacophore

N Pharrnacophore
Me,
OH

HN

R provides constraint
and hydrophobicity

Hydrophobic HO 0
groups
[(2S,3R)-TMT1]DPDPE

First generation of
non-peptide ligands
Figure 2.9. Conversion of (2S,3R)TMT1-c[DPDPElinto a nonpeptide peptide mimetic based on
-

topographical considerations.

tidomimetics has been around for over 20
years, since the discussions of Farmer (236).
There are many different ways in which the
term peptide mimetic or peptidomimetic has
been used, and the topic has been widely dis-
cussed from several different perspectives
(e.g., see Refs. 1, 8,9, 13-24,237-240). In the
context of the discussion here. the term non-
peptide peptide-mimetic is used to mean a bio-
active ligand with a nonpeptide scaffold that is
designed to mimic the pharmacophore of a
peptide ligand in three-dimensional space and
to have the same biological structure-activity
relationships as those of the peptide ligand.
Generally, this requires that there is a consid-
erable amount of insight into the conforma-
tional structure-biological activity relation-
ships of the peptide, including knowledge of
the three-dimensional topographical relation-
ship of key pharmacophore elements. In this
case (Fig. 2.9) this involved comprehensive
biophysical studies of the [TMT1]DPDPE an-
alogs (Table 2.16), including extensive NMR
studies, computational studies, molecular dy-
namic simulations, and molecular modeling
(241, 242). These studies led to a proposal for
the receptor pharmacophore in topographic
three-dimensional space. Several nonpeptide
scaffolds were considered and the 1,4-pipera-
zine was chosen (Table 2.17, I).In the initial
design of the peptide mimetics I (Table 2.171,
the major structural features of the peptide
pharmacophore were considered:
..The importance of the hydroxy-phenyl
group as a key pharmacophore element
(bothp-OH and m-OH groups were consid-
ered and evaluated, of which the m-OH
group gave the highest potency).
2. The requirement for a benzyl group as a
key pharmacophore element.
3. The distance between the two aromatic
group in three-dimensional space was a key
to delta opioid receptor selectivity of pep-
tide ligands.
4 Examples
Table 2.17 Nonpeptide Mimetics for Delta Opioid Receptors Based on Delta Opioid Peptides
Binding Affinity, IC,, (nM)
Structure p Receptor
"See Ref. 226.
bSeeRef. 227.
4. The need for a bulky R group for distin-
guishing peptide ligands for & versus
p-opioid receptors.
5. The requirement for a basic amine group
for 6-opioid agonist activity (in this case the
distance of the amine group relative to the
two aromatic groups was not optimized).
As can be seen in Table 2.17, increasing the
R group size from H to Me to Phe top-tBuPhe
(Ia, Ib,Ic, and Id, respectively)led to a steady
increase in binding affinity for the 6-opioid re-
ceptor; from about 6 f l to about 8 nit4 as
predicted (242). Most important, the selectiv-
ity for the &opioid receptor versus the p-opi-
oid receptor also increased very substantially
from nonselective to over 2000-fold selective
(Table 2.17), which actually is somewhat more
selective than DPDPE or [(2S,3R)TMT11
DPDPE (see Table 2.16 for comparison). In
the functional assays that make use of the
classical guinea pig ileum (GPI, for the p-re-
ceptor) and mouse vas deference (MVD, for
73
I1
6 Receptor Selectivity
the &receptor) compound Id was still highly
6-opioid receptor selective but was found to be
less potent in this assay than would be ex-
pected from its binding affinity. Subsequent
studies (Yamamura et al., unpublished) sug-
gested that ligand Id was a partial agonist. On
the other hand, structure-function studies
with further substituted derivatives of Id, and
studies at wild-type human gopioid receptor,
and a site-specific mutant receptor, demon-
strated that Id had properties of the peptide
ligand rather than that of other nonpeptide
ligands that had been discovered by evalua-
tion of structural libraries rather than by de
novo design. Nonetheless, the partial agonist
activity led us to design a number of further
analogs of I with modifications in the pipera-
zine ring. Starting with L-alanine, L-serine,
and L-phenylalanine, the analogs of I1 were
prepared (Table 2.17). Except for IIc, all of
these compounds had nanomolar binding to
&opioid receptors and were quite selective for
the 6-opioid receptor, but again in functional
74 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
assays they had lower potencies than what
would be anticipated from their binding affin-
ities (243). The importance of the two nitro-
gens also was examined (243, data not shown).
When the benzyl nitrogen was replaced by a
CH, group, the potency at the Sopioid recep-
tor decreased by nearly 3 orders of magnitude,
whereas when the other nitrogen was replaced
good potency at the S-opioid receptor was re-
tained, with some loss in selectivity.
These studies suggest that, although de
novo design of nonpeptide peptidomimetics
with high binding affinity and receptor selec-
tivity has an excellent chance for success,
there still is much to learn about those struc-
tural factors that are key for distinguishing
agonist from antagonist biological activity.
Agonist and antagonists for G-protein-cou-
pled receptors clearly have different struc-
ture-activity relationships, and in addition
may have certain dynamic structural require-
ments that are needed to bind to the receptor
to produce transduction for agonists and no
transduction for antagonist. In this regard, we
have recently shown (244), through the use of
a new spectroscopic method, coupled plasmon
waveguide resonance (CPWR or PWR) spec-
troscopy, which allows one for the first time to
examine changes in the structure of GPCRs in
membrane bilayers parallel and perpendicular
to the membrane bilayer normal, that when
delta opioid agonists and antagonists bind to
the human delta opioid receptor, the receptors
have different conformations, and that the
changes in conformation are consistent with
the differences in changes in structure for the
receptor that would be expected for transduc-
tion to occur or not to occur. The implication
of these findings suggests the need to be able
to evaluate those structural features critical
for agonist vs. antagonist activity at GPCRs in
both peptide and nonpeptide scaffolds. This
points again to the realization that there is
still no general predictable strategy in going
from agonist to antagonist ligands or vice
versa for GPCRs (245, 246), although there
are a number of approaches that have worked
in specific cases.
Returning to peptides for the delta opioid
receptor, a few other developments related to
high 6-opioid receptor selectivity should be
mentioned. The highly important TIPP and
TIPP[$] analogs of Schiller et al. already have
been discussed and a few of the most selective
analogs are given in Table 2.16. Of consider-
able interest were related dipeptides based on
the structure H-Tyr-Tic-OH, first reported by
Lazarus et al. (for a review, see Ref. 248). Par-
ticularly interesting were the analogs H-Dmt-
Tic-OH [DMT = (2s)-2',6'-dimethyltyrosine]
and the N,N1-dimethylanalog (N,N-Me,Dmt-
Tic-OH; Table 2.16). Originally, H-Dmt-
Tic-OH was reported to be exceptionally po-
tent and selective (250) but subsequent direct
comparison with TIPP analogs (249) indicates
that the most selective analogs in this series
are the tetrapeptide analogs in Table 2.16. At
the same time a more constrained series of
dipeptide analogs were prepared by Hruby et
al. (2511, in which all four isomers of TMT
were examined. As shown in Table 2.16, only
the (2S,3R) analog was found to be highly po-
tent and selective for the Sopioid receptor
(251). The (2S,3S) analog was much less po-
tent but retained good 6opioid receptor selec-
tivity. Both of the 2R analogs, (2R,3S) and
(2R,3R), were found to be essentially inactive
at both S and p-opioid receptors (251, data
not shown). Subsequently, based on extensive
second-messenger assays, it was shown that
H(2S,3R)TMT-Tic-OH was a highly potent
and selective (>6000-fold selective for the
delta versus mu receptor) inverse agonist at
the delta opioid receptor (252), providing an
important tool for evaluating the effects of in-
verse agonists in 6opioid receptor physiology
and pharmacology.
There are a few other approaches that have
led to highly potent and 6opioid receptor-se-
lective peptides. One of the most interesting
involves modification of DPDPE at the car-
boxyl-terminal. Of particular interest was the
discovery that modification of the t pen^ resi-
due with L-Cysor L-Pen (but not D-Cys or D-
Pen) and then adding an aromatic residue led
to analogs with unusual properties (Table
2.16) (253,254). As can be seen, the Phe6 com-
pounds are all as potent as or more potent (nM
to sub-nM) in binding affinity than DPDPE,
and have much higher selectivity, with the
Phe(pBr),Phe6analog having an IC,, value of
0.20 nM and a 21,000-fold selectivity. Even
more remarkable is the exceptional potency of
these compounds in the MVD (&receptor) in
, 5 Things to Come in Peptide and Peptidomimetic Design
vitro bioassay (data not shown, 253,254) with
the P h e ( ~ F ) ~ , P hanalog
e~ having an EC,,
value of 16 pm and a selectivity vs. the GPI
(preceptor) of 45,000 (254). The extraordi-
nary potency and selectivity of these com-
pounds can be attributed in part to their
greatly enhanced efficacy (255, 256) at &re-
ceptors. The structural and biochemical ori-
gins of such large increases in the efficacy of
signal transduction are still largely unknown,
but insight into their origins could provide im-
portant clues for the design of more efficacious
drugs (257).
Finally, various modifications of the deltor-
phins, which are naturally 8-opioid receptor-
selective ligands (see above), can lead to even
more potent and 6-opioid receptor-selective li-
gands. For example, Sasaki and Chiba (258)
prepared a series of C-terminally modified
peptide analogs related to the deltorphin, such
as the nBuG6- and (RS)secBuG6-constrained
analogs in Table 2.16, which are highly potent
and highly selective 6-opioid receptor ago-
nists. Misicka et al. (258) showed that use of
topographically constrained amino acids in
the Phe3 position, such as the (2S,3R)p-
MePhe3-containing analog in Table 2.16, can
provide a potent (IC,, = 2.4 nM) and highly
selective (>29,000) delta opioid receptor li-
gand. It also is possible to obtain good binding
h i t y and 6opioid receptor selectivity by
modifying the deltorphin sequence through
intermolecular cyclization such as the
[D-Pen2,~-Pen5]-deltorphin analog in Table
2.16 (257).
5 THINGS TO COME I N PEPTIDE AND
PEPTIDOMIMETIC DESIGN
The determination of the genome of humans
and many other animals and living systems
has opened up enormous opportunities for
peptide and peptidomimetic design, and for
the development of peptides and peptidomi-
metics as drugs, therapeutics, and diagnostic
reagents. Most cellular processes and system
activities, including those involving diseases,
are controlled or modulated by peptide-pro-
tein and/or protein-protein interactions. In
many protein-protein interactions fairly small
structural regions of one or both of the part-
ners are responsible for the biological effects.
Thus the development of peptides and pep-
tidomimetics that mimic these interactions
with high specificity can be expected to have
dramatic effects in their use as drugs of choice,
especially given that many peptides have very
low toxicities compared to those of current
drugs.
Some of these approaches include:
1. Continued development of conformational
constraints that can provide new peptide
and peptidomimetic motifs for design.
2. Continued development of topographical
constraints, especially in conjunction with
conformational constraints, will provide
new ways of evaluating peptide ligand-re-
ceptor/acceptor interactions.
3. Continued development of methods to sta-
bilize peptides against proteolyte degrada-
tion and to improve biodistribution.
4. Further development of new and more ro-
bust methods for peptide and peptide mi-
metic delivery and biodistribution.
5. Further development of assay methods, es-
pecially methods for evaluating peptide li-
gands in disease and pathological states as
a part of ligand design.
6. Further development of synthetic methods
for synthesis of large peptides and proteins
that will allow for more widespread use of
novel amino acid residues to explore pro-
tein function.
7. Continued development of peptide, pep-
toid, and PNA analogs and derivatives that
cross membrane barriers, target intercel-
lular receptor/acceptors, and enhance bio-
availability.
8. Development of a large variety of peptide
and peptidomimetic-based conjugates for a
variety of uses in diagnosis, drug delivery,
and treatment of diseases.
9. Continued investigation of novel scaffolds
that can mimic peptide secondary struc-
tures (+ and $ space), such as a-helices,
p-turns, p-sheets; peptide topographical
structures (chi space); and, most challeng-
ing, that can mimic protein conformational
changes such as a-helix to P-sheet transi-
tions.
76 Peptide and Protein Hormones, Peptide Neurotransmitters, and Therapeutic Agents
10. Continued development of computational
methods for evaluation of the conforma-
tional and dynamic properties of peptides
more quickly and more accurately.
It goes without saying that clearly there
will be a need for continued development of
biophysical methods that allow more rapid
and complete analysis of three-dimensional
structure and molecular dynamics. Methods
for studying the structural, conformational,
and dynamic properties in integral membrane
proteins such as GPCRs are critical, given that
more than 50%of current drugs use these re-
ceptors as a way of modulating biological func-
tions including disease. Of course, at the same
time biologists will develop better methods for
making use of genes, cells, tissues, and organs
for peptide and protein drug development.
Better models for evaluating disease states are
critically needed, and no doubt are high on the
agenda of many biologists and medical clini-
cians.
The ability of chemists and other physical
scientists to more effectively collaborate with
biological scientists and medical doctors is es-
sential for the future of mankind. Nearly all
problems facing us in this area require multi-
disciplinary approaches and ideas. The ability
to effectively communicate and collaborate to
solve problems in disease and diagnosis with
mutual respect and without any arrogance of
any field or discipline is critically required.
The very serious ethical and social issues that
modern science raises requires all scientists
and medical practitioners to take seriously
their own responsibility for what they invent
and how it will be used in society.
6 ACKNOWLEDGMENTS
We gratefully acknowledge the support of the
research from our laboratories by the U.S.
Public Health Service and NIDA. We particu-
larly thank Margie Colie for all her help in
preparing this chapter.
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CHAPTER THREE

Inhibitors of Gastric Acid

Secretion
SONIA ROBERTS
LUN M. MCDONALD
James Black Foundation
London, United Kingdom

Contents
1 Introduction, 86
2 Therapeutic Market, 86
3 Acid Secretion through the Ages, 88
4 How and from Where is Acid Produced?, 88
4.1 The Stomach, 88
4.2 Hormones and Neurotransmitters
Regulating the Secretion of Gastric Acid, 89
5 Physiology of Gastric Acid Secretion, 90
6 Disorders Associated with Elevated Secretion of
Gastric Acid, 91
6.1 Ulcer Disease, 91
6.1.1 NSAID-Associated Ulcer, 91
6.2 Zollinger-Ellison Syndrome, 92
6.3 Helicobacter Pylori (H. Pylori), 93
6.4 Reflux Esophagitis, 94
7 Test Assays for Studying Gastric Acid Inhibitors,
95
7.1 i n Vitro, 95
7.2 i n Viuo,96
8 Receptor-Mediated Processes that Regulate
Gastric Acid Secretion, 97
8.1 Ach-Muscarinic-Receptor-Mediated Acid
Secretion, 97 -
8.1.1 Structures and SAR of Anticholinergic
Agents, 97
8.2 Histamine Hz-Receptor-Mediated Acid
Secretion, 97
8.2.1 Histamine Receptor Classification, 98
8.2.2 H,-Receptor Antagonists, 99
8.2.3 Structures and SAR of Hz-Receptor
Antagonists, 99
8.2.4 Clinical Studies with Hz-Receptor
Burger's Medicinal Chemistry and Drug Discovery Antagonists, 101
Sixth Edition, Volume 4: Autocoids, Diagnostics, 8.2.5 Adverse Effects of Hz-Receptor
and Drugs from New Biology Antagonists, 101
Edited by Donald J. Abraham 8.2.6 The Need to Develop More Potent
ISBN 0-471-37030-4 O 2003John Wiley & Sons, Inc. Antisecretory Agents, 103

9 H+/K+-ATPase(Proton Pump)
Inhibition, 103
9.1 Structure of the Proton Pump, 103
9.2 Acid Secretion and the Proton Pump, 104
9.3 Mechanism of Action of Proton-Pump
Inhibitors, 104
9.4 Structure and SAR of the Proton-Pump
Inhibitors, 105
9.5 Profile of Proton-Pump Inhibition, 113
1 INTRODUCTION
The inhibition of gastric acid secretion is a key
therapeutic target for ulcer disease, gastro-
esophageal reflux disease (GERD), Zollinger-
Ellison syndrome (Z-E), and gastritis. Cur-
rently, this is achieved either by blocking the
acid-secretory effect of histamine (HA)
through the use of HA H2-receptor antago-
nists or by irreversibly binding to the Ht/K+-
ATPase with proton-pump inhibitors (PPIs),
to prevent the release of H+ ions from the pa-
rietal cell. These pharmacological approaches
are effective in alleviating ulcer disease, al-
though disease recurrence rates are high. The
discovery that ulcers are linked to Helicobac-
terpylori (H.pylori) infection has led to a new
therapeutic approach, in which eradication of
the bacteria is the principal target. In addition
to ulcer disease, H2-receptor antagonists and
PPIs are still effectively used for alleviating
the symptoms of gastritis, Z-E, and GERD.
The symptoms associated with each of these
conditions are similar; therefore correct diag-
nosis is essential. Recently, H2-receptor an-
tagonists have become available without pre-
scription, leading frequently to omission of the
critical diagnosis stage, and the self-adminis-
tration of these medications may mask the
symptoms of other diseases.
The aim of this review is to outline the
drugs that are currently available and the me-
dicinal chemistry that led to their discovery.
The physiological processes associated with
acid secretion and the mechanism of action of
inhibitors of this process are described. Cur-
rent research into inhibitors of gastric acid se-
cretion is aimed at producing a reversible PPI
or antagonist of receptor-mediated acid secre-
tion. The medicinal chemistry and pharmacol-
ogy of such approaches are considered.
Inhibitors of Gastric Acid Secretion
9.6 Clinical Studies with Proton-Pump
Inhibitors, 114
9.7 Adverse Effects of Proton-Pump Inhibitors,
115
10 Targets Under Investigation, 117
10.1 Reversible Proton-Pump Inhibitors, 117
10.2 CCKJGastrin-Receptor Antagonists, 119
11 Summary, 119
12 Acknowledgments,122
2 THERAPEUTIC MARKET
The therapy for gastric acid-related gastroin-
testinal disorders has evolved from nonspe-
cific gastro-protective agents to treatments di-
rected at specific sites regulating the secretion
of gastric acid. H2-receptor antagonists and
PPIs are currently the major therapies used to
inhibit the production of gastric acid. The dis-
covery that H. pylori infection was highly cor-
related with the presence of duodenal ulcer
and hypersecretion of gastric acid has intro-
duced an additional therapy that targets the
eradication of H. pylori. This combination
therapy of an antisecretory agent and an anti-
biotic has been shown to dramatically reduce
the number of patients in which ulcer forma-
tion recurs (1).The incidence of upper gastro-
intestinal disorders such as ulcer and GERD
shows an element of global variation. For ex-
ample, in Western countries duodenal ulcers
are more common, whereas in Eastern coun-
tries gastric ulcers predominate (2).These dif- '
ferences may be attributable to any of a num-
ber of factors, including diet and genetic
make-up. Therapeutic strategy also differs
from the East to West. In Western countries,
the conventional therapy for duodenal and
gastric ulcers is eradication of H. pylori
through the use of PPIs combined with an an-
tibiotic. In Japan, when eradication therapy is
used, a cytoprotective agent is also included.
Indeed, cytoprotective agents are used in
abundance, accounting in 1998 for approxi-
mately 50% of all prescribed treatments for
ulcer disease. In Japan, unlike the West, H,-
antagonists are commonly used for mainte-
nance therapy, with PPIs consisting of only
5% of the antisecretory therapy market. This
results from restrictions imposed by the Japa-
nese government that limit the prescribing of
2 Therapeutic Market
Table 3.1 Currently Prescribed Antisecretory Medicines in the UK
Generic Name Trade Name
Cimetidine Dyspamet
Zita
Tagamet
Dicyclomine hydrochloride Merbentyl
Esomeprazole Nexium
Farnotidine Pepcid
Hyoscine butylbromide Buscopan
Lansoprazole Zoton
Nizatidine Axid
Omeprazole Losec
Pantoprazole Protium
Ranitidine Zantac
Pylorid
Rabeprazole Pariet
PPIs to a maximum of 8 weeks, after which
patients are placed on H2-Rantagonists (2).
The incidence of ulcer disease appears to be
reducing worldwide, although whether this is
attributed to H. pylori eradication therapy is
unclear. However, the incidence of GERD is
increasing (3) and the control of this disorder
is a high profile pharmaceutical target. The
increased exposure of the esophageal epithe-
lium to gastric acid has been linked in some
cases to morphological changes of the normal
squamous epithelium into specialized intesti-
nal-like columnar epithelium. The formation
of this precancerous disease state called Bar-
rett's esophagus occurs in 15% of patients
with chronic GERD (4). It has been suggested
that reducing the exposure of this precancer-
ous tissue to further acid may prevent its sub-
equent malignant transformation.
Gastric acid-related diseases are common
d diverse, and their treatment should be de-
igned to alleviate the symptoms, while keep-
g the risk of adverse events to a minimum.
-receptor antagonists, although not long-
ting inhibitors of gastric acid, may be ade-
ate to treat gastritis, whereas PPIs may be
uired to totally inhibit gastric acid
ughout the day to effectively treat GERD
ients. Although PPIs have been available
ce the mid-1980s, GERD requires a much
gher therapeutic dose than that used for ul-
r disease. Currently, the long-term safety of
gh doses of PPIs is unknown.
A review of the leading pharmaceutical
products in 1985 placed the H,-receptor an-
Originator Chemical Class
Goldshield Hz-receptor antagonist
Eastern Hz-receptor antagonist
SmithKline Beecham H,-receptor antagonist
Florizel Anticholinergic
AstraZeneca Proton-pump inhibitor
M.S.D. Hz-receptor antagonist
Boehringer Ingelheim Anticholinergic
Wyeth Proton-pump inhibitor
Lily Hz-receptor antagonist
AstraZeneca Proton-pump inhibitor
Abbott Proton-pump inhibitor
Glaxo Wellcome Hz-receptor antagonist
Glaxo Wellcome H,-receptor antagonist
EisaiIJanssen Proton-pump inhibitor
tagonists Tagamet and Zantac as numbers 1
and 2, accounting for $894.2 and $565.8 mil-
lion, respectively. By 1990, Tagamet had been
usurped by Glaxo's direct competitor product
Zantac, which obtained the largest amount of
sales for 1990 ($2457.9 million) and 1995
($3523
.. million). The success of Zantac was as-
cribed to a major marketing effort that high-
lighted the antiandrogenic side effects of
Tagamet. By 1990, Zantac's growth had
slowed as H2-receptorantagonist therapy was
superseded by more potent therapies that in-
hibited the proton pump, such as Astra's
Losec (omeprazole). In 1995 when the patent
for Zantac expired, Losec became the third
biggest selling pharmaceutical ($2305 mil-
lion). By 1999 Losec had taken pole position as
the largest selling pharmaceutical product of
all ($5909 million), within an antiulcerant
class of therapeutics, valued at $12.9 billion.
The patent for omeprazole expired in 2001 but
AstraZeneca has counteracted this by market-
ing the (S)-enantiomer of omeprazole (esome-
prazole), which is reported to possess an im-
proved pharmacokinetic profile and potency
compared to that of the (R)-enantiomer (5).
(See Table 3.1 for currently prescribed antise-
cretory medicines in the UK.)
The decisions regarding the potential for de-
veloping other novel inhibitors of gastric acid
secretion are likely to be based on therapeutic
efficacy, specificity, lack of adverse effects, long-
term tolerability, and safety, as well as commer-
cial issues such as market share, patent expiry,
and generic competition (6).Self-medication has
88 Inhibitors of Gastric Acid Secretion

Table 3.2 Time-Related Discoveries with Respect to Gastric Acid Secretion

Year AuthorICompany Discovery
William Prout (7) Identification of HCl in gastric secretion of animals
William Beaumont Examination of acid secretion in humans
(182)
William Brodie (183) Performed first surgical vagotomy (dog)
Jean Pean (8) Performed first gastrectomy in humans
Ivan Pavlov (184) Examination of the neural regulation of gastric secretion
Leon Popielski (185) Found that HA stimulated gastric secretion in dogs
Andre Rapheal Laterjet Published on the physiological contribution of the vagus to
and Pierre acid secretion and motility
Wertheimer (186)
E. McCrea (187) Suggested that the vagus nerve might be implicated in
ulcer disease
Charles Code (188) Found increased HA-stimulated acid secretion induced
ulcer formation in animals
Lester Dragstedt (189) First truncal vagotomy in humans
Andrew Kay (190) Developed the HA test (acid secretory response produced
by HA in the presence of HI-receptor blockade)
I. Marks, Wilfred Card Use of HA test to determine parietal cell mass
(191)
James Black (69) Discovery of Hz-receptor and Hz-receptor antagonists
A. Ganser, J. Forte Discovery of H+/Kf -ATPase in oxyntic cells of the bullfrog
(110)
SmithKline & French Tagamet launched worldwide (Hz-receptor antagonist)
Glaxo Zantac launched (Hz-receptorantagonist)
AstraZeneca Omeprazole launched (PPI)
Glaxo Wellcome plc Ranitidine (Hz-receptorantagonist) generic forms
available
Takeda-Abbott Lansoprazole launched (PPI)
Yamanouchi (licensed Famotidine launched (Hz-receptorantagonist)
to Merck & Co.)
Eisai Co. (licensed to Rabeprazole launched (PPI)
Janssen
Pharmaceutica)
AstraZenceca Esome~razolelaunched (PPI)

been a major component of gastric acid inhibi-
tory therapies and therefore safety is of prime
importance. Both H,-receptor antagonists and
PPIs have been shown to be extremely safe and
well-tolerated drugs and future novel inhibitors
of gastric acid will have to show a comparable
level of safety.
3 AClD SECRETION THROUGH THE AGES
standing the role of the stomach in digestion
and diseases associated with hypersecretion of
acid. Modlin (8) provided an excellent review
of the discovery, hormones, mechanisms, and
drug discovery process linked to gastric acid
secretion. Table 3.2 summarizes these stages,
while focusing on the changing drug regime
used to treat disorders associated with the gas-
tric acid secretion.

The concept that the stomach secreted acid 4 HOW AND FROM WHERE IS AClD
and digestive enzymes was not fully appreci- PRODUCED?
ated until the nineteenth century, although
4.1 The Stomach
various hypotheses had been mooted since the
time of the ancient Greeks. The discovery of The stomach functions as a reservoir for in-
gastric acid (7) was the first step to under- gested food and as a primary site of digestion.
 4 How and from Where is Acid Produced?
I Esophagus
sphincter
Once food has entered the stomach, gastric
acid and enzymes are released and contraction
of the circular and longitudinal muscle of the
stomach wall produces thorough mixing of
its contents, aiding the breakdown of food be-
fore it enters the duodenum. The stomach has
both mechano- and chemosensitive receptors
within its structure and the presence of food
and its various components triggers specific
responses within the stomach. The stomach is
divided into four anatomical regions: the cor-
pus or body, fundus, antrum, and pylorus (Fig.
3.1). Enzymes and gastric acid are secreted
from glandular parts of the stomach located in
the antrum and fundic regions. The oxyntic
mucosa covers the fundic region and is the site
of acid secretion. Mucus cells line the mucosa
and invaginations of this epithelial layer form
the gastric glands, which consist of specialized
crypts that branch into specialized tubular
gland structures. Columnar epithelial cells
line the crypts, whereas numerous cell types
are found in the tubular glands (9).
4.2 Hormones and Neurotransmitters
Regulating the Secretion of Gastric Acid
The acid-secretingoxyntic cell, or parietal cell,
; is the main cell type in the gastric gland. Chief
, cells (pepsinogen-secreting cells), mucus cells,
I
and D-cells (somatostatin-secreting cells) are
also found within gastric glands. Somatosta-
tin, released from fundic D-cells, inhibits the
Figure 3.1. Anatomical regions of the
stomach.
release of acid secretion from the parietal cell.
These specialized cells are in close proximity
to parasympathic nerve endings and neural
inputs are able to regulate hormone release
(10). The release of HA from enterochromaf-
fin-like (ECL) cells can also be neurally medi-
ated and subsequently stimulates parietal
cells to secrete acid. Vagal nerve stimulation
initiates the release of gastrin from antral G-
cells, and acetylcholine (Ach) from postgangli-
onic, cholinergic, and muscarinic nerves,
which bind to their respective receptors on the
ECL-cell to release HA and subsequently acid
from parietal cells (Fig. 3.2).
Ach, HA, and gastrin stimulate acid secre-
tion by activating specific receptors on the ba-
solateral membrane of the parietal cell. Once
bound to the respective G-protein-coupled re-
ceptor, second-messenger systems are acti-
vated. Ach and gastrin activate phospholipase
C to catalyze the conversion of membrane-
bound phospholipids to diacylglycerol and ino-
sit01 triphosphate. The release of Ca2+ from
intracellular stores and the subsequent in-
crease in cytoplasmic Ca2+ activates Ht/K+-
ATPase (proton pump). The binding of HA to
the H,-receptor activates adenylate cyclase,
resulting in an increase in CAMP,which acti-
vates the proton pump (11).
Many peptides and neurotransmitters have
been identified as having a direct or indirect
effect on the parietal cell. Somatostatin, secre-
90 Inhibitors of Gastric Acid Secretior

HCI

\
Somatostatin
0
0
0
,
,
0

,,
,

Figure 3.2. Stimulation of acid secretion

from the parietal cell.

tin, and prostaglandin E reduce acid secretion
either indirectly, by inhibiting the release of
gastrin, or directly, by an action at the parietal
cell (12). Interleukins, vasoactive intestinal
peptide, cholecystokinin (CCK), calcitonin
gene-related peptide, oxyntomodulin, neuro-
tensin, adrenaline, and gastric inhibitory
polypeptide may inhibit parietal cell secretion
indirectly through the release of local soma-
tostatin. Peptide W, CCK, and 5-HT are
thought to influence acid secretion by modu-
-
lating neural tone to the stomach. The release
of nitric oxide release may also inhibit gastric
acid secretion.
In addition to mucus from goblet cells, bi-
carbonate ions are secreted from the stomach
to protect the gastric mucosa from gastric
acid. Bicarbonate secretion occurs when the
luminal pH is less than 2 and its release is
regulated both neuronally and hormonally.
5 PHYSIOLOGY OF GASTRIC ACID
SECRETION
Gastric acid secretion is stimulated by food-
related signals that stimulate the release of
acid from specialized cells within the stomach.
This secretory process has been divided into
three phases: cephalic, gastric, and intestinal,
with each phase leading to different amounts
of acid being secreted (Table 3.3).
Cephalic Phase. The sight, smell, taste, and
sensation of swallowing food causes central
stimulation of the vagus nerve leading to Ack
release from synapses within the fundic and
antral regions of the stomach. Direct stimula.
tion of the parietal cells within the fundus
and, to a lesser degree, the indirect stimula.
tion of HA release, through vagally stimulated
gastrin release from G-cells within the an
trum, causes the parietal cells to secrete acid
into the stomach.
Gastric Phase. Gastrin is the main media-
tor of acid secretion. Acid secretion occurs in
response to both the presence of nutrients and
the physical distension produced by food en.
tering the stomach. Distension-induced gas.
tric acid secretion, relative to the total amount
of acid released, is species dependent (human
= 20%; dog = 50%; rat = 38%). The chemical
constituents of a meal are the strongest stim-
ulant of gastrin release and acid output during
Table 3.3 Relative Acid Secretory Response
to Food
Percentage of Total
Phase of Acid Secretion Acid Secretion
Cephalic 50
Gastric
Distension 20
Chemical 25
Intestinal 5
6 Disorders Associated with Elevated Secretion of Gastric Acid
the gastric phase, with peptides and amino ac-
ids being greater stimulants of gastrin than
proteins, carbohydrates, and fats.
Intestinal Phase. Only a relatively small
ount of acid is secreted because of the nu-
erous inhibitory mechanisms stimulated by
e presence of nutrients within the intestinal
Inhibition of gastric acid secretion also oc-
s during the cephalic, gastric, and intesti-
phases. In the cephalic phase, the release
f various neuropeptides may contribute to
e inhibition of gastric acid secretion. Central
ection of neuropeptide Y (NPY), corticotro-
hin-releasing factor, bombesin, calcitonin,
itonin gene-related peptide, neurotensin,
erleukin 1, and prostaglandins have all
en shown to inhibit gastric acid secretion.
e exact mode of action of these peptides re-
sins to be elucidated, although they inhibit
tric acid secretion through vagal and sym-
athetic nerves. The hypothalamus appears to
important site of action of many peptide
itors of acid secretion. Of the peptides
udied so far, only NPY exerts both stimula-
ry and inhibitory effects on acid secretion
en injected into different hypothalamic
tes. In the gastric phase, increasing gastric
idity initiates a mechanism that turns off
tric acid secretion. Once the acidity of the
men has reached pH 2, gastrin release is in-
bited and therefore acid secretion is re-
ced. Stimulation of somatostatin release
D-cells in the antrum of the stomach in-
its the release of gastrin from G-cells and
us reduces gastric acid secretion. In patients
'th a duodenal ulcer this inhibitory process is
8s efficient, especially when intraluminal pH
s below 3. Eysselein et al. (13) also demon-
rated that patients with a duodenal ulcer ex-
ited higher rates of acid secretion yet a di-
nished capacity for inhibition of acid
cretion when low concentrations of peptone
re instilled intragastrically.
In the intestinal phase, inhibition of acid
cretion is produced by the presence of fat,
acid, or hyperosmolar solutions within the in-
testinal lumen. Fat, the most potent inhibitor,
was proposed to cause the release of inhibitory
substances from the intestine, and although
91
about the neural pathways that participate in
the fat-induced inhibitory reflex, although va-
gotomy does appear to reduce the inhibitory
effect of intestinal fat. This response is attrib-
uted to the altered sensitivity of parietal cells
to Ach in the absence of vagal tone. The re-
lease of CCK and somatostatin, in response to
intestinal acid, inhibits gastric acid secretion.
6 DISORDERS ASSOCIATED WITH
ELEVATED SECRETION OF GASTRIC ACID
6.1 Ulcer Disease
Peptic ulcers arise because of an imbalance of
acid-secretory mechanisms and mucosal-pro-
tective factors, and the rationale for their
treatment is aimed at restoring that balance.
The loss of balance between acid secretion and
mucosal-protective factors varies among pep-
tic ulcer types. In type I ulcers, which occur
high in the stomach, acid hypersecretion is not
necessarily evident, suggesting the impor-
tance of impaired mucosal-protective factors
in this clinical setting. Type I1 ulcers, in con-
trast, include gastric ulcers, distal antral
(prepyloric) ulcers, and duodenal ulcers. They
are associated with acid hypersecretion and
the impaired negative feedback effects of acid-
ification on gastrin release and on continued
acid secretion. The causes of gastric ulcers in-
clude H. pylori infection, nonsteroidal anti-in-
flammatory drugs (NSAIDs), environmental
factors, and malignancy. Duodenal ulcers can
result from hypersecretion of gastrin, which is
assessed by evaluating fasting gastrin levels in
patients unresponsive to other therapies for
duodenal ulcers.
6.1 .I NSAID-Associatkd Ulcer. NSAIDs
inhibit the production of prostaglandins, pros-
tacyclins, and thromboxanes from arachidonic
acid by covalently modifying the enzyme cy-
clooxygenase (COX) and irreversibly inhibit-
ing the ability of arachidonic acid to bind to
the active site on the enzyme. Chronic admin-
istration of NSAIDs has been linked to ulcer
disease, although there is no evidence that
they are the direct cause of ulcer formation. In
patients already diagnosed with ulcer disease,
chronic administration of NSAIDs was associ-
ated with a fourfold increase in the risk of ul-

cer-associated complications (14). These com-
plications, however, may remain undetected
because of the reduction in pain produced by
the inhibition of endogenous prostaglandins.
Inhibition of prostaglandin-derived gastropro-
tective properties such as gastric blood flow
and mucus production increases the exposure
of the mucosa to toxic agents. In an attempt to
restore the gastro-protective properties of the
prostaglandins, the synthetic prostaglandin
analog misoprostol may be coadministered.
However, therapies targeted solely at the sup-
pression of acid have been shown to be better
tolerated (15). Two isoforms of COX have been
identified: COX-1, a constitutively expressed
enzyme is found in many tissues, whereas
COX-2, a n inducible enzyme, is predomi-
nantly expressed at sites of inflammation. Se-
lective COX-2 inhibitors would therefore be
anticipated to reduce prostaglandin-depen-
dent inflammation, while leaving protective
gastric mucosal prostaglandin synthesis in-
tact. Ulcer healing involves the formation of
granulation tissue, reepithelialization, scar-
ring, and contraction of the ulcer base. Specific
inhibitors of COX-2, however, have been re-
ported to delay the healing of erosions in mice
and rats (16). Although COX-1 appears to pre-
dominate in human gastric mucosa, both
COX-1 and COX-2 are found at the rim of
gastric ulcers. Myofibroblasts, which are con-
sidered to play an important part in ulcer heal-
ing, express COX-2 and synthesize prostaglan-
dins when exposed to inflammatory stimuli.
Inhibition of COX-2 may thus retard ulcer
healing in human gastric mucosa (17).
Thromboxane, a major product of arachid-
onate metabolism, stimulates platelet aggre-
-gation and vasoconstriction. The chronic ad-
ministration of low doses of aspirin, a n
NSAID, as a prophylactic antithrombotic
agent, for patients with a history of heart dis-
ease, has been shown to double the incidence
of acute biopsy-induced bleeding in patients
(18). The antihemostatic properties of aspirin
on platelets may be linked with the high num-
ber of ulcer hemorrhages occurring in this pa-
tient subgroup (19). Aspirin inhibits both
COX-1 and COX-2 and as COX-2 selective in-
hibitors do not appear to alter platelet func-
tion they may act as a suitable antithrombotic
agents for high risk cardiovascular disease pa-
Inhibitors of Gastric Acid Secretion
tients with ulcer disease. Celecoxib, a selective
COX-2 inhibitor, appears to produce much
less gastroduodenal injury than standard
NSAIDs. In several animal studies, however,
selective COX-2 inhibitors have been shown to
interfere with healing of ulcers and to exacer-
bate inflammation (20). Current clinical trials
with COX-2 inhibitors have not directly exam-
ined hemorrhage and healing of ulcers in pa-
tients. However, given that a high percentage
of patients with NSAID-related gastric ulcers
do not experience disease-associated symp-
toms, the use of a COX-2 inhibitor, which
might worsen or delay the healing of ulcers in
a high risk patient population, might be dan-
gerous.
6.2 Zollinger-Ellison Syndrome
In this disease, a non-beta-cell tumor of the
pancreatic islets may produce gastrin in a
quantity sufficient to stimulate secretion of
gastric acid to life-threatening levels. The in-
troduction of Hz-receptor antagonists has
meant that total gastrectomy is no longer nec-
essary in treating these patients. Acid secre-
tion was initially controlled by high doses of
cimetidine or ranitidine and in some cases ad-
ditional surgery was carried out to remove re-
sectable tumors and reduce gastric acid secre-
tion by proximal gastric vagotomy. The
development of H'/K+-ATPase inhibitors,
such as omeprazole, has enabled adequate in-
hibition of gastrin-stimulated acid secretion to
be achieved for longer periods. Once- or twice-
daily dosing with omeprazole produces a pro-
found inhibition of gastric acid secretion;
therefore a vagotomy is no longer necessary.
Gastric ECL-cell carcinoids are rare events
that have been described in association with
pernicious anemia and Zollinger-Ellison syn-
drome. They usually relate to marked hyper-
gastrinemia, atrophic gastritis, or a genetic
defect, rather than the presence or absence of
acid. Regression or disappearance of ECL-cell
carcinoids may occur either spontaneously or
after removal of gastrin. H,-receptor therapy
may result in up to a twofold increase in
plasma gastrin, although no endocrine cell hy-
perplasia has been reported. Omeprazole
causes a two- to fourfold increase in plasma
gastrin and this results in hyperplasia in 7% of
patients (21).
6 Disorders Associated with Elevated Secretion of Gastric Acid
6.3 Helicobacter Pylori (H. Pylor~)
H, pylori infection occurs in approximately
40% ofthe population Over 40 years age and
most patients with peptic ulcer disease are in-
fected with H. pylori. Because ulcers recur in
patients who have undergone "successful" H.
pylori eradication therapy, infection may not
always be causative for the disease. Less than
5% of ulcer patients are H. pylori negative and
in H. pylori positive patients only 10% of ul-
cers recur after eradicating the infection.
Likely" causes of ulcer recurrence are consid-
ered to be reinfection, the use of ulcerogenic
drugs, and persistent gastric hypersecretion.
However, true reinfection is rare and it is
more likely" that the most common cause of H.
pylori recurrence is attributed to inadequate
eradication. The significant reduction in H.
pylori density produced by a combination of
antibiotic and antisecretory therapy may re-
duce the levels of infection to below detectable
levels (22). Rapid urease tests for H. pylori
have a sensitivity of only 80-90%. Therefore
histological examination is also used to con-
firm an initial noninvasive test result. Whole
blood or serum antibody testing are rapid, ac-
curate, and cost-effective tests for establishing
H. pylori status in rapid urease test-negative
patients. These less invasive techniques could
be used in place of endoscopy when the patient
has not previously been treated for H. pylori
(23).
In duodenal ulcer (DU) patients, H. pylori
infection causes inflammation of the antral
gastric mucosa, which is associated with an
elevation in gastric acid secretion. Gastritis in-
creases the release of the acid-stimulating an-
tral hormone gastrin and reduces the expres-
sion of the inhibitory peptide somatostatin.
Bacterial products and inflammatory cyto-
kines may produce these changes in endocrine
function. Gastritis involving the corpus tends
to decrease acid secretion, given that bacterial
products and cytokines inhibit parietal cells.
After eradication, gastrin levels are restored
to normal levels.
Gastric metaplasia is found in 90% of H.
pylori-infected DU patients and about 60% of
non-DU patients with increased acid secretory
levels. The induction of gastric acid causes se-
vere inflammation and increases the areas of
93
metaplasia within the gastric mucosa. Com-
bined eradication and acid-suppression ther-
apy produces greater reduction in gastric
rnetaplasia than either treatmentalone, indi-
cating that both acid secretion and H. pylori
infection contribute to ulcer formation. A
number of putative virulence factors for H.
pylori have been identified, including c a g ~ ,
vacA, and iceA. Although- disease-specific asso-
ciations have been claimed, there are now suf-
ficient data to state that none of these factors
is specific for any disease. The presence of a
functional c a g . pathogenicity island, whose
genes produce proinflammatory cytokines, in-
creases mucosal IL-8 and inflammation, but is
not predictive of the future development of a
disease. The hypothesis that iceA has disease
specificity has not been confirmed and vacA
genotyping has also failed to predict the dis-
ease identification. Virulence appears to be a
host-dependent factor. The pattern of gastritis
is frequently used to indicate the different H.
pylori-related diseases. The primary factors
responsible for the different patterns of gastri-
tis are suggested to be associated with envi-
ronmental factors, with the H. pylori strain
playing a minor role (24).
H. pylori infection is now proven to he a
risk factor for gastric cancer and the organism
was classified as a Group 1 carcinogen by the
International Agency for Research on Cancer
sponsored by the World Health Organization
in 1994 (25). This has strengthened the case
for H. pylori eradication to prevent gastric
cancer. However, there are growing concerns
that eradication may cause harm. In devel-
oped
- countries. an increase in the rate of can-
cers arising near the gastroesophageal junc-
tion may be linked to the disappearance of H.
pylori. The conundrum is to either eradicate to
avoid cancer of the distal stomach or leave it
and hence avoid cancer of the proximal stom-
ach or distal esophagus. More research is re-
quired to determine which patients should re-
ceive eradication therapy (26).
An increased incidence of GERD has been
linked to the decrease in H. pylori infection
produced by the current trend of eradication
therapy. H. pylori may have a protective role
by either reducing achlorhydria induced by
PPIs or by increasing the activity of PPIs by
increasing the formation of the active metab-

olite. The antisecretory effect of PPIs seems to
depend on the presence of the infection be-
cause eradication of H. pylori has negative
consequences on the efficacy of antisecretory
drugs (27).Several hypotheses have been sug-
gested to account for the reduction in efficacy
of PPIs in H. pylori-negative patients. Hyper-
secretory disorders may be associated with in-
creased expression of proton pumps on the
membrane of the parietal cell; therefore PPIs
would appear to be more effective in DU pa-
tients than in healthy controls. Other authors
have suggested that H. pylori infection may
inhibit proton-pump synthesis in the parietal
cell because the amount of H+/K+-ATPase
mRNA in the fundic gland mucosa was signif-
icantly increased in patients where H. pylori
had been eradicated (28). If a decrease in the
number of active proton pumps was the expla-
nation for the higher effectiveness of PPIs in
H. pylori-positive subjects, there should also
be a lower acid output and consequently
higher gastric pH in H.pylori-positive subjects
during baseline recordings. However, under
basal conditions, similar basal pH values are
recorded both before and after the cure of in-
fection. The most likely cause for the de-
creased effectiveness of PPIs is the production
of ammonia by H. pylori in infected patients.
Hz-receptor antagonists do not inhibit acid se-
cretion to the same degree as do PPIs; thus the
small amount of ammonia produced by H. py-
Lori would be unlikely to significantly aff'ect
the pH. However, the high gastric pH pro-
duced by PPI therapy would be influenced by
the reduction in ammonia produced by eradi-
cation of H. pylori. The reduction in H. pylori-
related gastritis produced during omeprazole
therapy has also been suggested to contribute
to the reduced activity of omeprazole after
eradication. However, histological improve-
ment of gastritis after a cure of H. pylori is a
slow process and the effect of reducing the ef-
fectiveness of omeprazole is rapid.
Although the usefulness of H. pylori eradi-
cation is still controversial, a randomized con-
trolled study has shown that patients, in
whom the organism has been eradicated, ben-
efit with regard to quality of life and there is
also a reduction in financial costs to the health
system (22).
Inhibitors of Gastric Acid Secretion
6.4 Reflux Esophagitis
Reflux esophagitis is a disorder of the defense
mechanisms at the esophageal junction, which
is caused by regurgitation of the gastric con-
tents, especially of gastric acid. GERD is asso-
ciated with decreased gastric emptying andlor
increased incidence of transient lower esoph-
ageal relaxation (T-LESR). Smoking and obe-
sity are factors that increase the incidence of
GERD-like symptoms such as heartburn,
belching, and bloating. Reflux has been ob-
served in humans and dogs but not in rodents.
Reflux can be subdivided into T-LESR. free
reflux, and stress reflux. H. pylori infection
does not necessarily correlate with GERD, al-
though a reduction in acid secretion reduces
the chances of reflux. The effectiveness of
PPIs is reduced in the absence of H. pylori
infection; therefore the majority of patients
with GERD require >20 mglday to provide
symptom relief and to heal the esophagitis
produced by gastric acid reflux. This dose is
much higher than that required to inhibit acid
secretion associated with DU disease. If PPI
therapy is stopped, then GERD patients ap-
pear to produce greater amounts of gastric
acid and their reflux is potentiated. It has been
-
reported that if the prevalence of H. pylori
continues to decline, then PPI consumption
will continue to increase for GERD (29). Ide-
ally, future therapy for GERD should be inde-
pendent of H. pylori status and additionally be
devoid of the acid-rebound effect produced
with PPI therapy.
GERD causes significant discomfort but is
not in itself life threatening. The incidence of
adenocarcinoma of the esophagus is increas-
ing in the United States (301, with almost
100% of cases occurring in patients with Bar-
rett's esophagus, a condition in which mucin-
secreting, metaplastic, columnar epithelium
replaces the normal squamous epithelium of
the esophagus (31). There is substantial evi-
dence that GERD may increase the incidence
of Barrett's esophagus. Fass reported that the
length of Barrett's esophagus tissue corre-
lated with the duration of esophageal acid
exposure (32). The use of antireflux medica-
tion in -patients with GERD leads to an im-
provement or alleviation of symptoms and
healing of mucosal inflammation. Antisecre-
 7 Test Assays for Studying Gastric Acid Inhibitors
tory agents have been used to reduce the ex-
posure of the esophagus to acid in Barrett's
esophagus. However, even high doses of PPIs
have not resulted in regression of Barrett's
mucosa. Not all GERD patients develop Bar-
rett's esophagus and the causative agents for
this progression to a precancerous state have
not been fully determined. The age of onset,
duration of symptoms, and complications of
GERD are markers of an increased risk of Bar-
rett's esophagus. In additon, the longer the
region of Barrett's mucosa, the higher the risk
for the development of dysplasia (33).
7 TEST ASSAYS FOR STUDYING
i GASTRIC ACID INHIBITORS
Gastric acid secretion occurs through recep-
tor-mediated or enzyme-mediated processes.
Drug dissociation constants can be deter-
mined from in vitro bioassays, allowing affin-
ity estimate comparisons to be made between
compounds. Functional evaluation can be
made using both in vitro and in vivo tech-
niques, with the latter models being able to
provide information on the pharmacokinetics
of drug candidates. Technological progress
made since the discovery of both Hz-receptor
antagonists and PPIs means that some meth-
ods used in the original publications can now
be replaced by cloned receptor systems.
7.1 In Vitro
Radioligand binding assays can be used to de-
termine the affinity of H,-receptor antago-
nists through the use of either cloned human
H2-receptorsexpressed in cultured cells or na-
tive H2-receptors in guinea pig cortex mem-
brane homogenates (34). Functional measure-
ments (e.g., HA-stimulated CAMPproduction)
can also be made in cell lines expressing either
native or cloned receptors (35).
The effect of gastric PPIs on Hf/Kf-
ATPase activity (ATP-induced phosphoryla-
tion) can be studied in vitro with partially pu-
rified Ht/K+-ATPase preparations from pig
gastric mucosa (36). Given that the enzyme
assay needs to be performed at neutral pH,
this system has been most effectively used to
study the mechanism of action of PPIs rather
than the structure-activity relationship (SAR)
of inhibitors. PPIs are prodrugs, requiring an
acid environment for conversion to the active
molecule (37), and it is the most chemically
labile molecules that appear particularly ac-
tive in this assay. Thus, misleading results
may be produced with regard to prediction of
clinical activity.
Gastric membrane vesicle preparations en-
riched with Hf/K+-ATPase have also been
used to examine PPIs. The inhibition of hydro-
gen ion transport by PPIs is measured by use
of the initial rate of acridine orange quenching
as an index of acidification. However, steady-
state acidification, as measured by aminopy-
rine accumulation, is inhibited with greater
potency and this is consistent with the accu-
mulation of PPIs in the intravesicular acidic
space (38).
To study the effects of PPIs a more "phys-
iological" situation is often used. Acid secre-
tion in vitro has been studied by use of isolated
parietal cells from guinea pigs (39), dogs (40),
and rabbits. Rabbit parietal cells and chief
A
cells, separated by density gradient centrifu-
gation, copurify with H+/Kt-ATPase. Stimu-
lants of acid secretion cause the accumulation
of radiolabeled aminopyrine, a weak base, into
the acid compartment of the parietal cell. At
pH 7 it can pass freely through biological
membranes in its un-ionized form but be-
comes trapped within the secretory cannal-
iculi because of ionization. The [l4Claminopy-
rine accumulation technique has been widely
used to study structure-activity relationships
of gastric acid inhibitors as well as to separate
the inhibitory effect of PPIs from that of re-
ceptor antagonists. H2-R antagonists inhibit
HA-stimulated acid secretion and aminopy-
rine uptake, whereas omeprazole, a PPI, in-
hibits both HA-stimulated and db-CAMP-stim-
dated acid secretion (41). [14ClAminopyrine
accumulation has also been used to examine
acid secretion in intact gastric glands from
rabbits (42) and humans (43).
A more recent cell-based in vitro assay in-
volves the use of the microphysiometer, a sen-
sitive extracellular pH sensor, which has been
A
used to measure luminal (or apical) Ht secre-
tion and basolateral release of OH- as well as
liberation of acidic metabolites in rabbit gas-
tric glands. Adenosine 3',5'-cyclic monophos-
phate stimulation produced a biphasic change

in the extracellular acidification rate (EAR)
that is attributed to an initial excess of baso-
laterally released OH- followed by delayed lu-
mind efflux of simultaneously produced H+.
The elevated EAR at steady state reflected lib-
eration of metabolic acid attributed to H+/Kf -
ATPase enzymatic activity. Both basolateral
OH- release and steady-state EAR were in-
hibited by the H+/K -ATPase inactivators
f
omeprazole (irreversible PPI) and SCH-28080
(reversible PPI) (44).
I n vitro assays used to examine gastric acid
secretion also include the use of isolated whole
stomach preparations from the rat (451,
mouse (46), and guinea pig (47). Whole, or
part, stomach preparations are placed in an
organ bath containing a buffered solution.
The stomach is perfused with an unbuffered
solution and changes in luminal pH are mon-
-
itored in response to stimulation of acid secre-
tion. Acid secretion is an oxygen-dependent
process, so this technique is limited to young
animals (45) with thin stomach walls or stom-
achs where the outer muscle laver " has been
removed. In older animals perfusion of the
vascular bed with oxygenated buffer is used to
maintain tissue viability (48). In these models,
acid secretion can be produced through either
direct or indirect stimulation of the parietal
cells using HA, gastrin, and Ach. It is also pos-
sible to examine the effect of drugs on neurally
mediated acid secretion by use of electrical-
field stimulation (49).
Rats and dogs are generally used to examine
gastric acid secretion in vivo. The anesthe-
tized rat model most commonly used is the
"Ghosh and Schild" rat (50). In this model,
intragastric pH is monitored by means of the
perfusion of an unbuffered solution through
the stomach, passing over a pH electrode upon
its exit. Drugs are generally administered
through intravenous, subcutaneous, or intra-
peritoneal routes. Oral administration would
require the flow of unbuffered solution to be
temporarily stopped, preventing the continu-
ous measurement of intragastric pH. The py-
lorus-ligated rat model, often termed the
"Shay rat" (511, combines drug administra-
tion to the conscious animal with gastric acid
collection under anesthesia (52). In this
Inhibitors of Gastric Acid Secretion
model. inhibitors of acid secretion are admin-
istered to the conscious rat through the oral,
intraperitoneal (i.p.), or subcutaneous (s.c.)
routes. After a specified time, during which
gastric emptying should have occurred, the rat
is anesthetized and the pyloric sphincter li-
gated to prevent further gastric emptying.
Acid secretion is then stimulated by HA, pen-
tagastrin, or Ach (usually s . ~or. i.p.) for a fixed
period of time, the animal euthanized, the
stomach excised, and the pH of its contents
determined. Both the Ghosh/Schild and Shay
rat (53,541models have been used in the eval-
uation of HA H, and CCK,/gastrin-receptor
antagonists, and PPIs.
Conscious rat and dog models involve the
use of surgically modified animals with a
chronic gastric fistula (55), enabling gastric
secretions to be collected and the pH deter-
mined by titration. The effect of drugs admin-
istered either orally or i.v. is examined on
basal and stimulated acid secretion. For oral
drug administration, the fistula must be
closed for a period of time to allow gastric emp-
tying. The surgical addition of an intraduode-
nal fistula allows drugs to be administered
"orally," while being able to continuously
monitor gastric pH. The brain is able to regu-
late the release of acid-secreting hormones, so
a surgical modification of the gastric fistula
was developed, the Heidenhain pouch model
(56). In this model, part of the stomach is di-
vided from the main body of the organ and
within the pouch the nerves are cut, so that
the pouch is isolated from the brain, but re-
tains the local blood supply of the stomach.
Hence, the pouch will secrete digestive juice in
response to a circulating hormone (gastrin)
produced by the main body of the stomach, but
not in resDonse to the extra stimulation from
>
the brain. Because the pouch is separate from
the body of the stomach, drugs and food can be
provided, whereas gastric juice, free of food
contamination, can be collected from the fis-
tula (57).
Significant species differences have been
demonstrated in gastric physiology and,
whereas studies in primates are rare, their
physiology has been shown to be similar to
that of humans (58). Primates are trained to
sit in a chair and, as in human studies, sam-
ples of gastric secretions can be taken through
8 Receptor-Mediated Processes that Regulate Gastric Acid Secretion
a nasogastric tube for pH analysis. Unlike the
dog, but in agreement with human studies,
primates produce significant basal and stimu-
lated acid secretion and the effect of inhibitors
of gastric acid secretion can be examined (59).
The more recent use of microdialysis
probes, implanted in the submucosa of the
acid-producing part of the rat stomach, en-
ables gastric acid secretion to be examined in
conscious animals without surgical modifica-
tion (60).
8 RECEPTOR-MEDIATED PROCESSES THAT
REGULATE GASTRIC ACID SECRETION
Gastric acid secretion from oxyntic cells is pro-
duced in response to stimulation of second-
messenger systems that trigger proton-pump
activation. Vagal nerve endings in the stom-
ach are stimulated by physicochemical factors
causing the release of Ach. Neurotransmitter
release from nerve endings that are in close
proximity to ECL and G-cells stimulates the
release of HA and gastrin, respectively. Each
of these processes can be disrupted to reduce
the amount gastric acid secretion. The ability
to selectively and effectively inhibit gastric
acid secretion determines the usefulness of
such therapies.
8.1 Ach-Muscarinic-Receptor-Mediated
Acid Secretion
Ach, released after vagal stimulation, binds to
muscarinic receptors present on both the acid-
secreting parietal cell and the HA-secreting
ECL-like cell (Fig. 3.3). Muscarinic receptors
stimulate the secretion of acid, pepsinogen,
and mucus in the gastric mucosa. Autoradio-
graphic techniques have shown that the M,
receptor is overexpressed in DU (61); there-
fore a selective M,-receptor antagonist may
provide a useful antisecretory therapy. Musca-
rink receptors are currently subdivided into
M,, M,, M,, M,, and M, (62). The receptors on
rat and rabbit parietal cells and human and
porcine gastric mucosa are of the M, subtype.
Pfeiffer (63) and Kajimura (64) found that
only the gene for the M, receptor subtype was
expressed in rabbit parietal cells. However,
the M,-receptor is also associated with smooth
97
muscle contraction and this may lead to unfa-
vorable side effects (65). (See Fig. 3.2.)
8.1.1 Structures and SAR of Anticholinergic
Agents. HA H,-receptor antagonists and PPIs
have largely superseded the use of muscarinic
anticholinergic agents in the control of acid
secretion. Muscarinic receptor heterogeneity
and wide tissue distribution, coupled with the
low subtype specificity of the early anticholin-
ergics such as propantheline, dicyclomine, and
hyoscine bromide, led to their being associated
with parasympathetic side effects such as dry
mouth, dizziness, constipation, and blurred
vision. Muscarinic antagonists, selective for
receptors located on the parietal cells, are con-
sidered more suitable candidates for anticho-
linergic agents in the control of acid secretion.
Moreover, the overexpression of the M, sub-
type in DU patients (61) has linked blockade
of this receptor subtype to decreased pain
through reduced duodenal motility. Pirenz-
epine, which in addition to helping distinguish
muscarinic receptor subtypes, was developed
as an antisecretory agent mainly on the basis
of its preferential inhibition of receptors on
the intramural ganglia of the stomach wall.
Compared to the H, antagonist ranitidine,
pirenzepine alone was less effective in the in-
hibition of acid secretion. The more potent de-
rivative telenzepine improved healing rates
(66); however, the increased incidence of side
effects with the antimuscarinic agents, has
failed to dislodge the H, antagonists as the
preferred antisecretory therapy. The effec-
tiveness of antimuscarinic therapy may be
limited, given that pirenzepine, a muscarinic
antagonist, inhibited peptone meal-stimu-
lated acid secretion by 39%, whereas raniti-
dine, an H,-receptor antagonist, produced
69% inhibition. In combination, however, the
acid-secretory response was almost com-
pletely inhibited (99%) (67). Although this
combination therapy is feasible, it is unlikely
to be used because of the side effects associ-
ated with muscarinic antagonists.
8.2 Histamine H,-Receptor-Mediated
Acid Secretion
Leon Popielski first identified HA as a major
stimulant of gastric acid secretion in 1916.

Dicyclomine Hyoscine butyl bromide
H3C
Telenzepine
Figure 3.3. Ach-receptor antagonists used in the control of acid secretion.
However, it was not until the synthesis of HA
H2-receptorantagonists that the role of HA in
parietal cell stimulation was fully recognized
(68, 69). An enlightening account of the Hz-
receptor antagonist discovery program at
SmithKline & French is provided in a review
by Duncan and Parsons (70). Originally, HA
was considered to be the final mediator of
secretagogue-stimulated gastric acid secre-
tion. However, although species dependent,
the hormone gastrin is also able to stimulate
gastric acid secretion, both directly (through
the oxyntic cell) and indirectly (through the
release of HA from the ECL cell). H2-receptor
antagonists inhibit both gastrin- and Ach-
stimulated gastric acid secretion, indicating
that the secretion of gastric acid can be medi-
ated by the indirect release of HA (71).
HA is synthesized from dietary histidine by
the enzyme histidine decarboxylase (HDC).
High levels of HDC activity are found in the
stomach, either in mucosal mast cells or in
ECL cells. Gastrin, Ach, and adrenaline inter-
act with their respective receptors on the
Inhibitors of Gastric Acid Secretion
Propantheline bromide
H3C
Pirenzepine
ECL-cell to trigger the release of HA from
storage granules located in ECL-like cells. HA,
by activation of the H2-receptor and subse-
quent elevation of CAMPlevels, stimulates the
parietal cells to secrete acid.
8.2.1 Histamine Receptor Classification.
HA receptors are currently subdivided into
HI, H,, H,, and H,. Gastric acid secretion
from the parietal cells is strongly stimulated
by HA, an action that is exerted through H2-
receptors and'is not inhibited by the classic
anti-HAS, which act a t HI-receptors. H,-
receptors are blocked by selective H2-receptor
antagonists, such as burimamide, cimetidine,
ranitidine, and famotidine. H,- and H,-recep-
tors are relatively newly discovered and their
physiological relevance is still being delin-
eated. H,-receptors regulate neurotransmit-
ter release from nerve terminals and, with
regard to acid secretion, H,-receptor stimula-
tion has been shown to inhibit pentagastrin-
stimulated gastric acid secretion in conscious
Heidenhain pouch dogs (72). The recently
8 Receptor-Mediated Processes that Regulate Gastric A,cid Secretion
cloned H,-receptors (73-76) have so far only
been identified in bone marrow and eosino-
phils, where their role is believed to be in the
regulation of the immune response.
8.2.2 H,-Receptor Antagonists. Hz-recep-
tors and the prototype Hz-receptor antagonist
burimamide were identified in a single paper
by Black et al. (69). Burimamide was reported
to inhibit both HA- and pentagastrin-stimu-
lated gastric acid secretion in rats, dogs, cats,
and humans. The potency of burimamide at
inhibiting gastric acid secretion far exceeded
that produced by anticholinergic drugs and
was devoid of apparent side effects. Burimam-
ide, however, had poor oral bioavailability and
was subsequently replaced by metiamide,
which was 10-fold more potent and its activity
could be detected after oral administration
(68).
Metiamide (400 mg) completely abolished
gastric acid secretion, whereas the maximum
tolerated dose of an anticholinergic, isoprop-
amide, produced only 35% inhibition of food-
stimulated acid secretion. In combination,
however, submaximal doses of the two antag-
onists provided a potent antisecretory combi-
nation (77). Metiamide went into clinical trial
in 1972 but was withdrawn because a small
number of patients developed agranulocytosis
during treatment. Initially, it was not certain
whether this toxicological problem was caused
by H2-receptorblockade affecting maturation
of the bone marrow, or whether the thiourea
moiety in the metiamide side chain had a di-
rect toxic effect on the cells (70). Cimetidine, a
molecule not containing a thiourea group, was
the third Hz-receptor antagonist to be tested
in humans. Cimetidine was similar to meti-
amide in its pharmacological profile, but did
not cause the agranulocytosis observed with
metiamide. Cimetidine was marketed in the
UK in 1976 under the trade name of Tagamet.
Cimetidine therapy led to a revolution in
the treatment of acid-peptic disorders, with
oral therapy being able to reduce the necessity
of surgical procedures. The discovery of this
landmark molecule caused a number of phar-
maceutical companies to be alerted to this
therapeutic area and target the discovery of a
"better" Hz-receptor antagonist. Ranitidine
was introduced by Glaxo in 1981 as a more
potent drug with a superior side-effect profile
and, backed by a skillful marketing campaign,
superseded cimetidine as the world's most
successful drug. Famotidine, synthesized by
Yamanouchi, was the third antagonist to be
registered and this remains the most potent
Hz antagonist available for clinical use. Two
other Hz-antagonists, nizatidine and roxati-
dine, are also commercially available, but they
seem to offer no improvement over the other
agents (6).
The clinically available H2-receptor antag-
onists are extremely safe. Toxicity problems,
however, were detected during the develop-
ment of other Hz-antagonists such as tioti-
dine, which induced glandular dilation and hy-
perplasia of the gastric mucosa. This
observation highlighted the necessity to exam-
ine the effects of antisecretory drugs on the
whole stomach, and later studies, either with
Hz-antagonists or proton-pump inhibitors,
produced similar hyperplastic properties
when administered for prolonged periods.
8.2.3 Structures and SAR of H,-Receptor
Antagonists. The success of cimetidine in the
control of gastric acid secretion in humans was
swiftly followed by a considerable effort to ob-
tain other Hz-receptor antagonists having a
superior profile, either in terms of potency or
duration of action. This process led to a wealth
of medicinal chemistry, which has been the
subject of a number of extensive and detailed
reviews (78,791 and to the evaluation of many
new compounds in clinical studies. The SAR
evolved from the imidazole-based antagonists
metiamide and cimetidine, and is illustrated
by representative compounds shown in Fig.
3.5. This led ultimately to the Hz-receptor an-
tagonists currently on'the market and shown
in Fig. 3.4.
Although the Hz-receptor antagonists span
a diverse range of structures, they can for the
most part be described by a general pharma-
cophore, that is, an aromatic or heterocyclic
ring, attached by a flexible, preferably
thioether chain, to a polar hydrogen-bonding
group. However, the breadth of chemical func-
tionality represented by each of these ele-
ments is testament to the rational approach
successfully used in their design. Thus, it was
largely by careful consideration of properties,
 Inhibitors of Gastric Acid Secretion

(c f N
N

H N*S H NHCH3
H~N-(,

4
s
Nx S q , N H

"J,
2

S02NH2
Cimetidine Farnotidine

Ranitidine Nizatidine
u

Roxatidine
Figure 3.4. Marketed Hz-receptorantagonists.

such as acidity, hydrophilicity, dipole mo-
ment, conformation, and geometry of the hy-
drogen-bonding group (go), that it was possi-
ble to retain Hz-receptor antagonism in a
variety of structures. For example, it was pos-
sible to replace the thiourea and cyanoguani-
dine groups of metiamide (81) and cimetidine
(82) with nitroguanidine and diaminonitro-
ethylene, respectively, which in this instance
proved to be effective bioisosteres. Other
groups such as guanidine (83), 2-amino pyri-
midin-6one (84),and 1,Cdiamino butene-2,3-
dione were less well tolerated, but they have
been used successfully in Hz-receptor antago-
nists, where additional changes have also been
made elsewhere in the molecule. Similarly, as-
sessment of the potential ionic and tautomeric
forms of the imidazole ring and the dynamics
of its interaction with the receptor led to the
suggestion that the neutral NH ' isomeric

Metiamide Tiotidine
0 0

Zolantidine W

Figure 3.5. Representative H,-receptor antagonists to illustrate SAR.

 Receptor-Mediated Processes that Regulate Gastric Acid Secretion
rm is the preferred bioactive isomer (85,861.
his prompted the preparation of many ana-
ich either substituents such as
oro (87) and methylthio (88) were intro-
ere the imidazole ring was re-
laced by other heterocycles.
Although 2-pyridine, 2-thiazole, and %so-
all successfully used for this pur-
se, the most marked increase in potency
th respect to cimetidine was achieved by re-
idazole ring by a guanidinethi-
obtain tiotidine (891, as well as
otidine (go), in which the cyanoguanidine
group had additionally been replaced by the
more hydrophilic sulfamoylamidine substitu-
ent. Moreover, with greater basicity now re-
ocyclic substituent than that in
heterocyclic ring itself, the importance of
s characteristic in the latter substituent di-
nished. The presence of a furan ring as the
heterocyclic component in ranitidine is consis-
tent with this view (91), and a similar struc-
tural motif is also present in nizatidine (92).
The dimethylaminomethyl-substituted furan
of ranitidine evolved further to include piper-
idinylmethyl-substituted phenyl ethers, rep-
resented by roxatidine (93). An acetyl acet-
was used as the hydrogen-
tuent in this case and higher
as achieved when this group was re-
the bulkier and more lipophilic ami-
iazole ring, as in zolantidine (94). A
similar trend toward higher affinity arising
from such groups is observed in oxmetidine
(95)and lupitidine (96), which can be consid-
ered to be more potent analogs of cimetidine
and ranitidine, respectively.
In general, the introduction of branching to
the linking chain afforded less potent com-
pounds, whereas replacement by an aromatic
linker was successful in the case of tiotidine
analogs but only when the attached groups
had a meta disposition (89). The thioether link
has been retained in most Hz antagonists be-
cause substitution of the sulfur atom by other
heteroatoms or by methylene has been detri-
mental to affinity at the receptor. This has
been attributed to a requirement to main-
tain an intramolecular hydrogen bond, be-
tween the polar termini, observed in crystal
structures (97) and during infrared studies
(98) of Hz antagonists and which the
thioether link uniquely helps to maintain.
However, whether this is a necessary re-
quirement for biological activity remains
unclear.
8.2.4 Clinical Studies with H,-Receptor An-
tagonists. HA Hz-receptor antagonists are po-
tent and selective inhibitors of gastric acid se-
cretion, with numerous clinical studies to
support their effectiveness in ulcer disease.
Table 3.4 outlines the clinical effectiveness
and pharmacokinetic profile of Hz-receptor
antagonists currently available in the UK.
Cimetidine, ranitidine, famotidine, and
nizatidine exhibit similar profiles of absorp-
tion, distribution, and elimination. Each of the
Hz-receptor antagonists exhibits classical
competitive drug-receptor interactions, with
Schild slope parameters not significantly dif-
ferent from unity (99). The affinity of each of
these drugs for the Hz-receptor is reflected in
their effectivenessin inhibiting gastric acid se-
cretion (100). Famotidine is the most potent
Hz-R antagonist, being 20-50 times more po-
tent than cimetidine and 6-10 times more po-
tent than ranitidine (101) and nizatidine.
Each of these drugs is rapidly absorbed after
oral administration, with peak plasma concen-
trations being achieved within 3 h of dosing.
Oral bioavailability for these drugs ranges
from 43% to 90%. With the exception of niza-
tidine, the bioavailability of Hz-receptor an-
tagonists is reduced because of extensive first-
pass hepatic metabolism. Only minimum
plasma protein binding occurs (130%)and all
of the Hz-antagonists are eliminated quite
rapidly, with a terminal half-life of 1to 3 h and
a total body clearance of 24-48 L/h. Elimina-
tion is mainly attributable to renal excretion,
with renal clearances ranging from 13.8 to 30
L/h. Given that the values for renal clearance
greatly exceed the glomerular filtration rate
(6-7.2 L/h), it is apparent that renal tubular
secretion plays an important role in this pro-
cess (102).
8.2.5 Adverse Effects of Hz-Receptor An-
tagonists. The Hz-receptor antagonists are
generally extremely safe drugs, with few ad-
verse effects being reported. Cimetidine was
shown to possess antiandrogen properties in a
small number of patients; however, this effect
Table 3.4 An Overview of the Clinically Marketed H,-R Antagonists: Affinity, Clinical Pharmacology, and Pharmacokinetic Profilea
Plasma Volume of Plasma
In vitro Half-life Distribution Protein
Drng PKB Clinical Pharmacology: Effect on Gastric Acid Secretion Oral Absorption (h) (L kg-l) Binding Excretion Metabolism

Cimetidine 6.1 (193) Normal 0.8-1.2 (194) 1325% (194) Kidneys: i.v., -70% Oxidation to the sulfoxide
unchanged (195) (195)
i.v. p.0. p.0.
= 2 pmo1 L-' O.Sl.0 giday; 71% 0.8 giday; 55% 1.6 glday;
stimulant; decrease in 24 h, 67% decrease in 24 h,
pentagastrin, 40 pg mean H' (174) mean H+ activity
kg-' h-' (195) (174)
Famotidine 7.8 (193) ICSO= 4.3 pg kgg-lh-' 5 mg; 40% inhibition 40-80 mglday; improved 1.1-1.4 (194) 20% (194) Kidneys: i.v., 70% Oxidation to the sulfoxide
stimulant
pentagastrin, 0.1 pg
kg-' h-' (196)
5 mg inhibition to
cimetidine
(300 mg) (197)
healing rates;
different dosing
regimes examined.
-
unchanged (199) p.o.,
40% unchanged;
remainder in bile or
(194)

Effect on gastric acid metabolized (194)

secretion was not
examined (198)
Ranitidine 6.7 (193) Ranitidine (150 mg) PGstimulated acid 1.82 (203) 15% (202) Kidneys: i.v., 69.4 ? Hepatic, some
more potent than secretion, 40 mg; 42% 6.1%; p.o., 26.7 + 7.2% enterohepatic recycling
cimetidine (200 mg) 80 mg; 69% nocturnal (203) (202)
(200) acid secretion, 80 mg;
50% (201)
Nizatidine 30 mg 57%; 100 mg Nizatidine (300 mg) was 1.1 (194) 30% (194) Kidneys (204) -35% major metabolites:
73%; 300 mg 90%; signifcantly shorter- N-2 mono des
75mg= . lasting than both methylnizatidine (-7%
inhibition to ranitidine (300 mg) of dose), nizatidine N-2
cimetidine (300 and farnotidine (40 oxide (-5%);nizatidine
mg) (205) mg) (100); nizatidine sulfoxide, minor
(300 mg) produced metabolite (207)
similar healing rates
to those of ranitidine
(300 mg) (206)

"Abbreviations: i.v., intravenous administration; p.o., oral administration; i.d., intraduodenal administration; normal, healthy human volunteers; DU, duodenal ulcer patients.
9 H+/K+-ATPase (Proton Pump) Inhibition
was not detected with other H2-receptor an-
tagonists (103). Cimetidine also has high af-
finity for the cytochrome P450 system,
whereas ranitidine has intermediate affinity
and famotidine and nizatidine have little to no
interaction with this pathway (104). Inhibi-
tion of this enzyme system can decrease the
oxidative metabolism of other drugs, resulting
in decreased clearance. H2-receptor antago-
nists may therefore adversely affect the clear-
ance of agents that have a limited therapeutic
window (e.g., warfarin, theophylline, and
phenytoin).
8.2.6 The Need to Develop More Potent An-
tisecretory Agents. The discovery and develop-
ment of Hz-receptor antagonists was the be-
ginning of a novel therapeutic approach to
diseases associated with the hypersecretion of
gastric acid. The inhibition of acid secretion at
the level of the parietal cell, by inhibiting the
activation of HA released from ECL-cells, was
the beginning of cellular-based antisecretory
therapy. During the 19809,Hz-antagonists be-
came first-line therapy in peptic ulcer disease
and negated the necessity of surgery for a
large number of patients.
It soon became apparent that H2-receptor
antagonist therapy had some drawbacks. In a
small but significant number of patients, the
disease was resistant or recurred after maxi-
mal H2-antagonist therapy (105). In another
small group of patients, H2-receptor antago-
nists were particularly poor when it came to
inhibiting the nocturnal secretion of gastric
acid (106). The limitation of this therapy re-
sulted from the actions of other mediators,
such as Ach or gastrin, acting directly on the
parietal cell, in stimulating the secretion of
gastric acid. In some patients, tolerance to H2-
antagonist therapy also appeared to develop
when long-term treatment was necessary
(107, 108). A further problem was that acid
rebound (elevated acid secretory response) oc-
curred after cessation of H2therapy (109). The
upregulation of other hormonal mechanisms,
alterations in signal transduction, or receptor
upregulation were hypotheses proposed to ac-
count for both the decreased effectiveness and
acid rebound, which was produced by Hz-an-
tagonist therapy.
The discovery of the proton pump (110) and
the elucidation of its function (111)were the
first steps toward the discovery of another
novel antisecretory therapy. In 1977, a year
after the launch of cimetidine, Astra reported
on the first PPI, H83/88, a benzimidazole de-
rivative that noncompetitively inhibited both
receptor (HA)-mediated and nonreceptor
(CAMP)-mediatedacid secretion from isolated
gastric mucosa (112).
9 H+/K+-ATPase (PROTON PUMP)
INHIBITION
The enzyme H /Kf -ATPase, or proton pump,
f
regulates the final stage in the cellular cascade
that terminates in the secretion of gastric acid.
Acid secretion is dependent on the cellular lo-
cation of the enzyme and the close proximity
of a potassium chloride efflux pathway. When
the parietal cell transforms from a resting to a
stimulated state, cytoskeletal rearrangement
occurs and H+/K+-ATPase relocates to the
apical plasma membrane. Potassium and chlo-
ride ions move across the apical cell mem-
brane together with secreted protons. Potas-
sium is recycled, whereas the hydrochloric
acid of gastric juice is formed by chloride ions
together with secreted protons (Fig. 3.6).
Neuronal, hormonal, and enzymic path-
ways influence the secretion of gastric acid.
The diversity of therapies available for treat-
ing hypersecretory disorders appear to have
developed with these targets in mind (i.e., va-
gotomy, Hz-, and muscarinic-receptor antago-
nists and, more recently, PPIs). The most po-
tent inhibitor of gastric acid secretion is
produced by inhibiting Hf/Kt-ATPase.
9.1 Structure of the Proton Pump
The H+/Kf-ATPase enzyme, present in tubu-
lovesicular and canalicular membranes of the
gastric parietal cell, consists of two subunits, a
114-kDa a-subunit and a 34-kDa p-subunit.
The a-subunit has been shown to contain 10
transmembrane helices, with the p-subunit
possessing only a single transmembrane helix
(113). The a-subunit carries out the catalytic
and transport functions of the enzyme be-
cause it contains both ATP and cation binding
sites; it also contains the sequences responsi-
ble for apical membrane localization. When

Figure 3.6. Activation of the proton pump in the parietal cell.
the parietal cell transforms from an active to
resting state, the heavily glycosylated p-sub-
unit is required for endocytic retrieval of the
H+/Kt-ATPase from the canalicular mem-
branes. This subunit may also contribute to
proper folding and membrane localization of
the enzyme (114). Ht/Kt-ATPase belongs to
the family of Pz-type ATPases and has homol-
ogy with other members of this family such as
Na+/K+-ATPase (65% homology) and Ca2+-
ATPases (23% homology).
9.2 Acid Secretion and the Proton Pump
When the resting parietal cell is stimulated by
acid secretagogues, the tubulovesicles are
transformed into the secretory canaliculus.
The parietal cell has the largest mitochondria1
content of any mammalian cell (-34% of cell
volume) and the ATP generated by this is
mainly used for acid secretion. Hydrolysis of
ATP results in a conformational change in the
protein that mediates the electroneutral ex-
change of intracellular Ht and extracellular
K+. The pump is activated only when it is as-
sociated with a potassium chloride pathway in
the canalicular membrane (Fig. 3.6). This al-
lows potassium chloride efflux into the extra-
cytoplasmic space and thus results in the se-
cretion of HC1 at the expense of ATP
lnhibitors of Gastric Acid Secretion
Parietal cell
KC1 transport
pathway
hydrolysis. The stimulation of hydrogen ion
secretion by the ATPase leaves behind an
equivalent number of hydroxide ions. These
are converted to H C O , by the enzyme car-
bonic anhydrase, which is closely associated
with the secretory membrane. The activity of
the pump is determined by the access of K+ to
the extracytoplasmic surface of the pump. In
the absence of K' on this surface of the pump,
the pump cycle stops at the level of the phos-
phoenzyme.
In recent studies, mice lacking a functional
Ht/K+ATPase (achieved by ablation of the
Hf/K+ATPase a-subunit) were found to pos-
sess severe disturbances in the secretory
membranes of the parietal cell, metaplasia of
the gastric mucosa, achlorhydria, and hyper-
gastrinemia (115). Hf /Kt-ATPase therefore
forms a critical component of the ion-trans-
port system mediating acid secretion in the
stomach.
9.3 Mechanism of Action of
Proton-Pump lnhibitors
PPIs are both more potent and of longer dura-
tion than Hz-receptor antagonists and there-
fore are frequently the drug of choice for the
treatment of diseases associated with the se-
cretion of gastric acid. PPIs inhibit gastric acid
9 H+/K+-ATPase (Proton Pump) Inhibition
secretion by inhibiting the enzyme H+/Kf -
ATPase, which is located on the luminal sur-
face of gastric parietal cells. Currently pre-
scribed PPIs are substituted benzimidazole-
based structures. When administered at
neutral pH, these weak bases are chemically
stable, lipid-soluble, and have no effect on gas-
tric acid secretion. The inactive PPI diffuses
from the bloodstream into the parietal cells
and subsequently into the acid environment of
the secretory canaliculi, where it rearranges
to form a sulfenic acid in equilibrium with a
sulfenamide. Either chemical entity is then
able to interact covalently with thiol groups at
cysteine residues located on the luminal sur-
face of the a-subunit of the H+/K+-ATPase.
This covalent binding results in specific and
essentially irreversible inactivation of the en-
zyme, leading to long-lasting inhibition of gas-
tric acid secretion (Figs. 3.7 and 3.8). Such a
profile is thus ideally suited to once-daily ad-
ministration in the management of acid-pep-
tic disorders.
Like omeprazole, the other commercially
available PPIs, lansoprazole, rabeprazole,
pantoprazole, and esomeprazole are inactive
prodrugs that are activated in the acid envi-
ronment of the gastric glands. This means
that all these agents are best administered in a
formulation that includes an enteric coating.
Subtle differences in the inhibitory effects of
these compounds on H+ transport and acid
secretion, however, have been reported. These
have been attributed to binding of the drug to
different cysteine residues within the proton
pump. Unlike omeprazole and lansoprazole,
which bind to two (cyst413 and cys892) and
three (cys813, cys892, and cys321) cysteine
residues, respectively (116), rabeprazole is re-
ported to bind to a single cysteine residue lo-
cated at position 322 between transmembrane
domain H3 and the lumen. This interaction
does not appear to result in a conformational
change of the enzyme, whereas omeprazole
stabilizes the conformation of the enzyme in
the dephosphorylated state, or so-called E2
form (117).
9.4 Structure and SAR of the
Proton-Pump Inhibitors
In 1973 workers at AB Haessle in Sweden,
identified timoprazole (118) as one of the
first well-defined inhibitors of the newly - dis-
covered gastric proton pump. This compound
stemmed from efforts to separate the toxicity
and acid-inhibitory properties of 2-pyridyl-
thioacteamide (CMN131). Removal of the
thioamide group was considered to be the
most likely solution to the toxicity of CMN131,
which prompted the preparation of sulfur-
containing heterocycles, as well as imidazo-
line- and benzimidazole-linked sulfides. How-
ever, it was the corresponding S-oxide analogs
of the latter compounds that proved the more
potent; it thus became clear that the mecha-
nism of action of timoprazole was distinct
from that of Hz-receptor antagonism. Timo-
prazole was subsequently followed by the
more potent derivatives picoprazole in 1976
and omeprazole in 1979 (119). The three main
structural features of omeprazole (i.e., the
substituted pyridine ring; the substituted
benzimidazole; and the methylsulfinyl linking
group, by which these two ring systems are
attached to one another) are essential, either
in generating the active form from its inactive
prodrug precursor or in binding irreversibly
with the H+/Kf -ATPase enzyme. For this rea-
son, compounds inhibiting acid secretion by
this mechanism and lacking one or more of
these features are scarce. Moreover, for irre-
versible proton-pump inhibitors to achieve se-
lective biological activity, their mechanism of
action demands that they have relatively high
chemical stability around neutral pH, but be
readily activated at low pH. This chemical pro-
file also influences attendant issues such as
synthesis, formulation, and storage, and be-
cause biological activity of this class of com-
pounds often correlates with chemical lability,
not all compounds, which achieve potent in-
hibitory behavior in vitro,are viable drug can-
didates because they are inherently too chem-
ically unstable.
The gastric proton-pump inhibitors cur-
rently available (Fig. 3.10) all retain the same
key chemical features present in omeprazole,
indicating that the structural requirements to
achieve irreversible inhibition of the gastric
ATPase enzyme are precisely defined. The
clinical properties of this latter group of drugs
is discussed more fully in section 9.6, whereas
the remainder of this section focuses on other
candidates currently or previously under de-
 Inhibitors of Gastric Acid Secretion

I I I
Orneprazole %
, I
\ I I
\ I
I Oesophagus I

orne~razolein acid
environment of
. - - pariental cell

pH 7.4 - Via bloodstream

/
/
I

Figure 3.7. Conversion of omeprazole to its active state within an acidic environment. Oral admin-
istration of enterically coated omeprazole ensures maximal activation occurs within the parietal cell.

velopment. As discussed above, the potency of
irreversible proton-pump inhibitors is a time-
and pH-dependent property, making compar-
ison of their in vitro potency difficult, given
the wide range of assay conditions employed
(Table 3.5 and Fig. 3.9). Where possible, their
potency relative to omeprazole, measured un-
der the same assay conditions, is indicated.
However, notwithstanding differences in the
stimulant used, comparison of their in vivo
9 H+/K+-ATPase (Proton Pump) Inhibition

r -
(b) H3C,0

Figure 3.8. Acid-catalyzed activation of omeprazole to form the active pyridinium sulfenamide or
sulfenic acid. Only one isomer of intermediates (a) and (b) is shown.

potency, as judged by inhibition of gastric acid
secretion, is in many respects more reliable
because interspecies variations are minimized
and the data obtained are also unconfounded
by differences in pH.
The same chemical features retained in the
marketed compounds lansoprazole (120), pan-
toprazole (1211, and rabeprazole (122) are re-
tained in disuprazole (54) TY-11345 (123), Ro-
18-5364 (1241, and SKF 95601 (125). The
benzimidazole group of omeprazole has been
replaced by other heterocycles and its activity
retained, with the methoxyimidazopyridine
compound tenatoprazole (126) being the most
advanced. Similarly, the fused benzene ring of
the benzimidazole group has been substituted
by a thiophene ring in the thieno[3,4-dlimida-
zole-based compounds saviprazole (127) and
8-1924 (128); however, examples of the corre-
sponding thieno[2,3-dlimidazole isomers (I),
aIso described by Hoechst, are much less
effective, most likely because of their greater
chemical stability. Although many of the com-
pounds lack substituents on the benzimid-
azole ring, the presence of electron-donating
groups at the 5-position, such as methoxy
(omeprazole), SKF 95601, OPC-22575 (129),
S-337 (128), pyrrol-1-yl (IY-81149) (130), and
difluoromethoxy (pantoprazole),also provided
an optimum balance of chemical stability and
reactivity. In fact, the presence of electron-
withdrawing substituents in this position,
such as nitro, methylsulfinyl, and trifluor-
methyl, increases the basicity of the benzimid-
mole ring to the point where the behavior of
these compounds is dominated by activation at
neutral pH, resultingin compounds having poor
chemical stability and limited practical value
(131).
In contrast, increasing the nucleophilic
character of the pyridine ring, by the incorpo-
ration of electron-donating substituents, en-
Table 3.5 Biological Activity of Representative Irreversible PPIs
In Vitro In Vivo
Inhibition of Gastric Acid
Secretion in Rat by Enteral

aN&i'
Ic50, a(Ic5O1 fl, Administration, ED,,, mg kg-'
Structure omeprazole) (ED,,, mg kg-', omeprazole) Ref.
20 (50)a 2-5 (2.5)d 54
/ N
H

H3C S7
CH3

Disuprazole (Upjohn)

TY-11345 (Tao Eiyo KK)

cl'
c-
SKF 95601 (SmithKline & French)
szN+(v
Tenatoprazole (Mitsubishi-Tokyo Pharm. Inc.)

H
N

0CH2CF2CF2CF3

Saviprazole (Hoechst AG)

OPC-22575 (Otsuka Phdrm. Co. Ltd.)

IY-81149 (11-Yang Pharm. Ind. Co. Ltd.)

Table 3.5 (Continued)
In Vitro In Vivo
Inhibition of Gastric Acid
Secretion in Rat by Enteral
Ic50, f l (Ic50, f l , Administration, ED5,, mg kg-'
Structure omeprazole) (ED,,, mg kg-', omeprazole) Ref.

Leminoprazole (Nippon Chemipharm. Co. Ltd.)

(2) (Roussel Morishita Co. Ltd.)

 YJA 20379-4 (Yungjin Pharm Co. Ltd.)

-L
GYKI-34655 (Egis Gyogyszergyar RT)
d
d
"Canine gastric (H+/Kt) ATPase.
*Rabbit gastric (H+/K+) ATPase.
'Porcine gastric (Ht/K+) ATPase.
dShay rat.
=Ghoshand Schild rat.
fIntravenous administration.
112
Timoprazole (Astra AB)
H3C
S-1924 (Hoechst)
Ro-18-5364 (Roche holding AG)
Figure 3.9. Structures of representative irreversible PPIs to illustrate SAR.
hances the rate of attack of the C-2 position of
the benzimidazole group and thereby pro-
motes the acid-catalyzed rearrangement to
the active species. This electronic feature is
-
present in omeprazole (3,5-dimethyl-4-me-
thoxy) and pantoprazole (2,3-dimethoxy),
whereas it is combined with increased lipophilic
character in 4-fluoroalkyl benzimidazole-substi-
tuted compounds, such as lansoprazole (2,2,2-
trifluoroethyloxy) and saviprazole (2,2,3,3,4,4-
heptafluorobutyloxy). The 3-methoxypropow
substituent present on the pyridyl group of ra-
-
beprazole has a particularly strong electron-do-
nating character, but the resulting enhance-
ment in reactivity in this case is mitigated by
formulation as its sodium salt, so as to increase
chemical stability. Similarly, the 3-chloro sub-
stituent is used to similar effect in the case of the
4-aminopyridyl-substituted compound SKI?
95601 (131).
As an alternative to the 2-pyridyl group of
omeprazole, compounds containing a substi-
tuted benzene ring in this position, made suf-
ficiently electron rich by the presence of a
2-amino group, display potent inhibition. Al-
though other examples of this type are known
(S-3337, OPC-22575), leminoprazole (132) is
Inhibitors of Gastric Acid Secretion
HN
S-337 (Hoechst) L ~ ~ 3
H3C 0CH3
the most advanced clinically and displays an
enhanced duration of action relative to that of
omeprazole. Similarly, the electron-rich-sub-
stituted pyrimidin-2-yl group (2)has also been
used in place of the 2-pyridylmethyl moiety in
compounds described by Roussel-Morishita
(133).
AD-9161 represents the most potent of a
novel but mechanistically similar class of irre-
versible H+/K+-ATPase inhibitors (134).
Compounds of this type were designed based
on the finding that the oxoisothiazolo~5,4-
blpyridine (3) displayed potent inhibition of
ATPase in vitro but failed to achieve inhibi-
tion of gastric acid secretion in vivo (135). This
disparity was ascribed to the rapid and prefer-
ential attachment to and inactivation of (31, by
thiol groups located outside the region of the
ATPase enzyme in the parietal cells. AD-9161
is considerably more stable than (3)and dis-
plays potency comparable to that of omepra-
zole both in vitro and in vivo.
Although all the proton-pump inhibitors
described so far are prodrugs that rely on the
participation of a sulfoxide group to effect
their activation, related sulfide analogs are
also known. Because compounds of this class,
9 H+/K+-ATPase (Proton Pump) lnhibition
such as YJA-20379-4 (136) and GYKI-34655
(137), would first need to be oxidized to the
sulfoxide before activation, they are consider-
ably less potent than omeprazole in vitro, but
in vivo they are at least as effective in models
of gastric acid secretion and ulceration.
In an effort to obtain a second-generation,
irreversible proton-pump inhibitor having a
superior profile, renewed interest has recently
focused on the relative potency of the optical
isomers of omeprazole and other benzimida-
zold-yl2-pyridylthiomethylS-oxides. The op-
tical activity arises from chirality at the sulfur
atom. One aim was to reduce liver clearance
and thereby minimize variations in interpa-
tient bioavailability and efficacy. The enanti-
omers of omeprazole were long expected to be
of equivalent potency to one another and to
omeprazole, given that acid-catalyzed conver-
sion to the nonchiral active forms would be
expected to occur at the same rate. Use of a
single enantiomer was also an unattractive
approach, given that, once separated, the en-
antiomers slowly racemized, although it was
subsequently confirmed that they displayed
similar behavior in uitro (138). It was only
with the availability of larger quantities of the
respective enantiomers (139) and their stabi-
lization by formulation as alkaline salts, that
their relative potency i n viuo could be rigor-
ously investigated. In rats, the enantiomers
displayed distinct profiles in their potency and
bioavailability because of selectivity in their
rates of metabolism. Subsequently, in human
studies, the relative potency of the enanti-
omers was found to be reversed, with the S-
isomer now being the more potent, because of
the presence of the CYP2C19 cytochrome
P450 metabolizing enzyme for which the R-
isomer is a substrate. This is an enzyme that is
absent from rats (140).
Based on its superior bioavailability in hu-
man studies (141), esomeprazole, the S-enan-
tiomer of omeprazole, has now been launched
as a successor, with the aim of providing a
higher level of management in acid-related
disorders. Single-enantiomer versions of lan-
soprazole [(S)-lansoprazole]and pantoprazole
[(-)-pantoprazole] (142) are currently in the
early stages of clinical development.
9.5 Profile of Proton-Pump lnhibition
Omeprazole is a potent and irreversible inhib-
itor of Hf /Kt-ATPase. Subsequently devel-
oped compounds (lansoprazole, pantoprazole,
esomperazole, and rabeprazole) have a similar
mechanism of action, although small differ-
ences in bioavailability, potency, and metabo-
lism have been reported.
Comparative studies with lansoprazole and
pantoprazole suggest that they have a potency
similar to that of omeprazole (143). Rabepra-
zole is readily converted to the active drug
form and has a more rapid onset of effect than
that of omeprazole in both in vitro and in vivo
studies. In the rat, there is evidence to suggest
that rabeprazole may have a slightly shorter
duration of action than that of other PPIs.
Compared with lansoprazole, the onset of ac-
tion of rabeprazole was faster and its duration
of action was shorter, as determined by mea-
suring acid output and microsomal enzyme ac-
tivity (144). However, in another study in
dogs, the rate of recovery of acid output was
similar when rabeprazole was compared with
omeprazole (145). The rate of activation of
PPIs is pH dependent, with rabeprazole being
fully activated at pH 5.0, whereas omeprazole
and lansoprazole are only partially activated.
These different pH selectivities may have an
impact on drug interactions and safety (146).
Rabeprazole is claimed to possess properties
different from those of the other PPIs, with
regard to the number of cysteine binding sites;
its effect on the conformation and partial re-
activity of the enzyme; the rate of acid activa-
tion (122) and thus onset of action (147); anti-
secretory potency i n vivo (148); and the
kinetics of reversal by scavenger mechanisms
(117). Unlike omeprazole and lansoprazole,
rabeprazole is reported to bind to just one cys-
teine residue located at position 322 between
transmembrane domain H3 and the lumen.
This interaction appears not to result in a con-
formational change in the enzyme. Conver-
sion of rabeprazole into a reactive intermedi-
ate also appears to occur more rapidly than for
other PPIs, which results in a faster onset of
action and greater potency in isolated cell sys-
tems. Compared with omeprazole, the rate of
reversal by glutathione is more rapid and the
compound has a shorter duration of action, as

Orneprazole (Astra AB)
Rabeprazole (Eisai Co. Ltd)
Esorneprazole (Astra AB)
Figure 3.10. Marketed irreversible PPIs.
measured in isolated gastric glands and some
animal models. Compared with lansoprazole,
the onset of action of rabeprazole was faster
and its duration of action was shorter, as de-
termined by measuring acid output and micro-
somal enzyme activity (144).
9.6 Clinical Studies with
Proton-Pump Inhibitors
PPIs are currently the most rapid, potent, and
long-lasting treatment for hyperacidity disor-
ders. Omeprazole was first marketed in 1988
and still remains the drug of choice for many
patients. Like omeprazole, the majority of
subsequently marketed PPIs, pantoprazole,
lansoprazole, and esomperazole, bind irre-
versibly to the proton pump. Acid secretion
can be restored only through endogenous syn-
thesis of new Ht/K+-ATPase, which has a
half-life of production of approximately 50 h
(116). Rabeprazole, however, is converted
more rapidly into its activated forms and
dissociates more readily from the H'/K'-
ATPase, resulting in a faster rate of inhibition
and a shorter duration of action. This property
of rabeprazole is most likely linked to its acti-
lnhibitors of Gastric Acid Secretion
Pantoprazole (Byk Gulden)
Lansoprazole (Takeda Industries Ltd.)
\
0CH3
vation within a more neutral pH range than
that of any of the other PPIs, with pantopra-
zole being the most stable (143). (See Fig.
3.10.)
The plasma half-life of the PPIs is rela-
tively short (-1 h) and accumulation is un-
likely to occur, even when clearance is signifi-
cantly reduced by renal impairment. The oral
bioavailability of esomeprazole is lower than
that of the other PPIs, with lansoprazole being
the most orally bioavailable (149). All PPIs are
highly protein bound and are metabolized in
the liver.
Although PPIS are currently the most po-
tent inhibitors of gastric acid secretion avail-
able, omeprazole has been reported to produce
interindividual variations in its ability to inhibit
gastric acid secretion. Recently, one of the isoen-
zymes of cytochrome P450, CYP2C19, which is
integral to the degradation of PPIs in the liver,
was shown to have two genetic phenotypes: ex-
tensive metabolizers and poor metabolizers.
Variations in the phenotypes of CYP2C19 have
been shown to affect the acid-suppressing ef-
fects of omeprazole, although the effectiveness
of rabeprazole was not affected by CYP2C19 ge-
9 H+/K+-ATPase (Proton Pump) Inhibition
notype (150).The potency of omeprazole, lanso-
prazole, and pantaoprazole was dependent on
metabolism by the enzyme CYP2C19 and, in
subjects not expressing this genotype, the effec-
tiveness of these PPIs was increased sign&
cantly. The genetically polymorphic CYP2C19
enzyme is absent from 3% of Caucasians and
20% of Asians.
It is difficult to make direct comparisons of
the relative activity of each of the PPIs, given
the different doses and dosing regimes used in
clinical studies . However, for most PPIs, ef-
fectiveness has been compared to that of the
prototype PPI, omeprazole. Table 3.6 outlines
the results obtained in some of these clinical
studies as well as the pharmacokinetic profile
determined for each of the PPIs marketed in
the UK.Clinical studies for Hz-receptor an-
tagonists tended to measure effectiveness re-
lated to inhibition of acid secretion. In con-
trast, healing rates were generally the
parameter of choice for PPIs. The PPIs are
clearly more potent than the Hz-receptor an-
tagonists, with clinically used doses being at
least 15 times lower than those of Hz-receptor
antagonists used in the treatment of DU (151).
The pharmacokinetics and acid suppression
produced by omeprazole, pantoprazole, lanso-
prazole, and rabeprazole from numerous clin-
ical studies were recently reviewed (143). The
authors concluded that these PPIs were of
ivalent potency, although rabeprazole and
soprazole displayed a more rapid onset of
ion. As part of the triple therapy used for
adication of H. pylori, each of the PPIs was
ly effective.
Until recently, all PPIs were marketed as
ures of enantiomers. However, the devel-
ment of esomeprazole has prompted numer-
studies to test its therapeutic benefit over
at of existing PPIs. Within the last 2 years
more than 40 publications have reported
udies involving the use of esomeprazole. Es-
eprazole has an improved pharmacokinetic
ofde relative to that of omeprazole: esome-
ole (20 mg per day for 5 days) had a 70%
gher area under the plasma concentration-
e curve than that of omeprazole (20 mg per
for 5 days). The S-isomer of omeprazole
meprazole) was found to undergo less me-
lism by CYP2C19 than the R-isomer in
human liver, this decreased metabolism ac-
counting for esomeprazole's improved phar-
macokinetic profile (152). The other advan-
tages of esomeprazole over the existing PPIs
are also related to its reduced metabolism by
cytochrome P450 enzymes in the liver. With
most other PPIs, drug interactions are of con-
siderable importance, especially in situations
where decreased metabolism is likely, for ex-
ample, where the CYP2C19 enzyme is absent
or there is hepatic or renal impairment. Fur-
thermore, because esomeprazole undergoes
less hepatic metabolism compared with that of
omeprazole and other PPIs, there is less inter-
patient variability in the metabolic profile, the
presence or absence of CYP2C19 being of less
importance. This makes the drug easier to
manage (153).
Although esomeprazole is a potent inhibi-
tor of acid secretion, clinical trials comparing
its effectiveness to that of other agents have
tended to use disparate doses for each PPI ex-
amined (154). Kromer et al. considered that
there was no pharmacodynamic argument in
favor of single-enantiomeric formulations of
any PPI (146). Moreover, potential pharmaco-
kinetic differences between the enantiomers
seem to be of little if any importance in the
patient. A clinical study comparing the cost
effectiveness of esomeprazole and omeprazole
in the acute treatment of GERD found that
esomeprazole 40 mg once daily was more cost
effective than omeprazole 20 mg once daily
(155). This cost-cutting marketing approach
used by AstraZeneca may encourage the pre-
scribing of esomeprazole over omeprazole or
its imminent generic substitutes.
9.7 Adverse Effects of Proton-Pump
Inhibitors
Headache is one of the most frequently re-
ported adverse events in clinical trials where
PPIs have been examined (frequency
1.3-8.8%). Patients with headache also had a
significant incidence of diarrhea, nausea, and
dizziness. A discontinuation of PPI therapy
resulted in a cessation or reduction of the
headache in 80.0% (20 of 25) (156).
Possible risks of prolonged treatment with
PPIs had been thought to include hypergas-
trinemia (1571, atrophic gastritis, and enteric
infections. However, no data have established
any true risk to patients, despite many years
Table 3.6 An Overview of the Clinically Marketed PPI Inhibitors; Affinity, Clinical Pharmacology, and Pharmacokinetic Profile
Volume of Plasma
In Vitro O d Plasma Distribution Protein
h~ PKR Clinical Pharmacology: Effect on Gastric Acid Secretion Absorption Half-Life (h) (L kg-') Binding Excretion Metabolism
Omeprazole 9 (208) Normal 54% (209) Urine:feces Hepatic hydmxylation
4:l (210) through CYP2C19
(211,212)
1.v. p.0. p.0.
40 mg, fewer inhibition of: basal 10,20, and 30 mg/day
Pre-oP acid, 30 mg, 66%; for 1 week caused a
patients with 60 mg, 92%; PG 37,90, and 97%
pH < 2.5 and stimulated acid, decrease of 24-h
a volume of 30 mg, 71%; 60 mg, intragastric acidity
25 mL (213) 95%; 7-day (215)
treatment, 30 mg
and 60 mg, 100%
inhibition (214)
Pantoprazole 8.6 (208) 80, 120 mg Maximal inhibition Healing rate, 20 mg, 77% (217) Renal, Hepatic hydmxylation
maximal with 60 mg; 40 mg, 58%; 40 mg, 89%; -80% through CYPZCN
inhibition for pH<3for8h, 80 mg, 82% (217); (217) (212); subsequently
up to 21 h; equieffectivewith 4-weeks treatment; metabolized by
onset of omeprazole (217) healing rate: sulfotransferase
action < 1h omeprazole, 20 mg, (218)
(216) 77%; pantroprazole,
40 mg, 88%
Lansoprazole 9 (208) - Maximal inhibition Dosebeding r a t e 30 ss% (221) Biliary, Hepatic hydroxylation
with 230 mg (219) mg/86%; omeprazole renal through CYP2C19
20 mg/82% (220) (222) (212)
Rabeprazole - 10, 20,30, and 40 mg, 20 mg in conjunction 52% (224) Renal, Hepatic hydmxylation
dose-dependent with antibiotic -90% through CYP2C19
inhibition of acid eradication rates: (224) and CYP3A4 (143)
secretion (223) omeprazole, 69%;
rabeprazole, 84%

Esomeprazole - 9 20 mg Day 1,46%,

Day 5,90% (140)
(224)
20 mg in conjunction
with antihiotic
20 mg, 68%;
40 mg,
Urine:feces
4:1(210)
Hepatic hydmxylation;
mainly metabolized
eradicationhealing 89% (226) by CYP3A4,
rate: omeprazole, compared to R-
88192%; esomeprazole omeprazole, which is
86191% (225) almost completely
metabolized through
CYP2C19 (140)
10 Targets Under Investigation
of experience with these agents. PPIs are re-
markably safe and well tolerated (158).
10 TARGETS UNDER INVESTIGATION
10.1 Reversible Proton-Pump Inhibitors
The effectiveness of clinically" available PPIs
relies on the number of active pumps at any
one time and the recovery of pumps after bio-
synthesis. The prolonged suppression of gas-
tric acid secretion produced by both Hz-recep-
tor antagonists and PPIs produces extended
periods of hypergastrinemia, which has been
associated with the formation of precancerous
changes in human gastric mucosa and gastric
carcinoids in long-term animal studies. In
fact, the development of omeprazole, a proton-
pump inhibitor, was delayed because high
doses were shown to induce ECL-cell hyper-
plasia in rodents. However, Astra disputed sci-
entifically that this would occur in humans
and omeprazole was eventually marketed in
1988.
Nonetheless, some research efforts are cur-
rently targeted at obtaining reversible PPIs,
so-called acid pump antagonists (APAs).
These are proposed to offer distinct advan-
tages over both the irreversible proton-pump
inhibitors and HA Hz-receptor antagonists
(159).Acting at the final stage of gastric acid
secretion, molecules of this type have the po-
tential to combine profound inhibition of gas-
tric acid secretion elicited by all stimuli and
the dose flexibility available with Hz-receptor
antagonists. Several companies have pro-
gressed APAs into development, although cur-
rently none has progressed beyond phase I11
clinical trials.
From the medicinal chemistry point of
view, the imidazopyridine-based compound
SCH 28080 was the prototype of the reversible
proton-pump inhibitor (160). As early as 1983,
it had been suggested that the antisecretory
effect of this compound was directly mediated
by the gastric proton pump, and this has been
further demonstrated by its ability to antago-
nize the binding of the irreversible proton-
pump inhibitor omeprazole (161). The marked
liver toxicity of this compound necessitated
follow-up compounds, of which SCH 32651
(162) is an example. Yamanouchi and Shin-
Nippon have also described the properties of
structurally similar compounds, respectively
YM-020 (163) and SPI-447 (161). Develop-
ment of SPI-447 is ongoing and it has been
shown that the compound has no effect on
Na+/Kt-ATPase activity. The mechanism of
SPI-447 is thought to be SH-group indepen-
dent, Kt-competitive, and highly specific
against gastric Ht/Kf-ATPase (161) (Fig.
3.11).
Another program to obtain a reversible
proton-pump inhibitor came from workers at
SmithKline & French, who selected the sub-
stituted quinoline compound SK&F 96067 as
an early clinical candidate (164). SKF 96067 is
a reversible inhibitor of the Ht/K+-ATPase
protein of the parietal cell (164). In clinical
trials SKF 96067 was found to be a more po-
tent inhibitor of gastric acid secretion than the
Hz-receptor antagonist ranitidine. This com-
pound reached Phase I11 clinical trials but has
now been discontinued (165). The compound
was followed by SKF 97574 (159) that, al-
though of similar potency to that of SKF
96067, displayed a significantly longer dura-
tion of action in vivo.
As an alternative strategy to escape the
drawbacks of the initial leads, the quinoline
ring was retained but constrained in a pyrrolo-
quinoline ring system as in SKF 96356 (166).
This ring system has also formed the basis of
compounds described by the Korea Research
Institute of Chemical Technology [AU-006
(1671, AU-461 (168), and DBM-819 (169)l.
DBM-819 displays potency comparable to that
of omeprazole in models of gastric acid secre-
tion and ulceration (169).
A tetrahydroisoqunoline-based compound,
YH-1885, discovered by Yuhan, is currently
one of the most clinically advanced reversible
proton-pump inhibitors (170). It is now being
codeveloped with GlaxoSmithKline for stom-
ach ulcers and gastroesophageal reflux dis-
ease. Clinical data on YH-1885 have demon-
strated that it is safe and well tolerated when
administered as a single dose (60 to 300 mg) or
multiple doses (150 to 300 mg) to healthy vol-
unteers. The compound significantly in-
creased gastric pH and increased the fraction
of time above pH 3 at doses above 150 mg.
During multiple dosing, YH-1885 exhibited a
reversible mode of action with no significant
 l I
0,
CH2C6H5
H 3 C ~ 0 YM-020
SCH 28080 SCH 32651 CH3 (Yamanouchi)
(Schering-Plough) (Schering-Plough)

SPl-447 I I
(Shin Nippon) 0CH3 SKF 96067 HOW' SKF 97574
(SmithKline and French) (SmithKline and French)

I H I H
0CF3
SKF 96356 F3c-0 AU-461 AU-006
(SmithKline and French) (Korea lnst. of (Korea lnst. of
Chem. Technology) Chem. Technology)
H3C0

H
DBM-819 YH-1885
(Korea lnst. of Chem. (Yukan) (4) Banyu Pharm Co.
Technology)

T-776 H2N/
(Tanabe)

Figure 3.11. Representative reversible PPIs to illustrate SAR.

accumulation (171).YH-1885recently entered
phase I clinical trials for the treatment of
Banyu Pharmaceutical Co. has disclosed
the properties of proton-pump inhibitors such
as (41, which were obtained by chemical mod-
ification of the structure of omeprazole, but
which are reversible in their interaction with
the ATPase enzyme (172). Tanabe, also, has
described a proton-pump inhibitor (T-776),
which contains many of the structural ele-
ments found in the irreversible inhibitors, but
which has been shown to possess a reversible
mechanism of action (1731.
10.2 CCKJGastrin-Receptor Antagonists
Gastrin is the only gut peptide hormone re-
leased from the stomach that mediates gastric
acid secretion. Gastrin-stimulated release of
gastric acid is produced either by direct stim-
ulation of CCKJgastrin-receptors on the pari-
etal cell or, in some species, indirectly after
CCK.Jgastrin-receptor-mediated HA release
from ECL cells. HA activates H2-receptors on
the parietal cell to stimulate gastric acid secre-
ion. The regulation of gastrin and HA-stimu-
edgastric acid secretion are key therapeutic
gets in controlling hyperacidity disorders.
ion is regulated by several hor-
ones and the inhibition of acid secretion
and PPIs has a positive feedback
ffect on the release of gastrin (174, 175).
A number of chemically diverse CCK2/gas-
agonists have been examined
als as antisecretory agents or in-
itors of paniclanxiety attacks. To date,
has been marketed (Table 3.7).
KJgastin-receptor antagonists, including
ose compounds in preclinical development
well as those that have progressed to hu-
e subject of a recent review
The importance of gastrin in development
investigated through the use of hormone
d receptor knockout mice. Gastrin and gas-
-receptor knockout mice are viable, fertile,
hout any gross abnormalities.
ough gastrin expresses trophic properties
hypergastrinemia (e.g., perni-
s anemia), no general atrophy of the gas-
observed in knockout mice.
ere was, however, a decrease in the acid-
secretory capacity of both mutants. Basal acid
secretion was abolished and parietal cells were
unresponsive to stimulation by HA, gastrin, or
Ach. In gastrin knockout mice the ECL cells
contained fewer secretory granules and were
located closer to the base of the gastric glands
compared to that in normal, litter-mate con-
trol mice, an indicator of poor ECL cell activ-
ity. In both the gastrin and gastrin-receptor
knockout mice, the number of parietal cells
was reduced (177).
Current gastrin-receptor antagonists
(CCG-receptor antagonists) inhibit the ac-
tion of amidated gastrin, although they do not
interact with the site at which glycine-ex-
tended gastrin binds (178). The gly-gastrin re-
ceptor has not been cloned, although there is
strong functional evidence that its stimulation
by glycine-extended gastrin is a link to prolif-
erative behavior (179). The Aphton Corpora-
tion has developed an immunogen, gastrim-
mune, which is composed of the amino-
terminal portion of G17 linked to a diphtheria
toxoid. This immunogen stimulates the pro-
duction of antibodies to both the amidated and
glycine-extended forms of gastrin-17 and has
been demonstrated to reduce the growth of
implanted colon tumors in rats and mice
(180). After encouraging results in phase I1
clinical studies (1811, gastrimmune, in combi-
nation with chemotherapy, entered phase 111
clinical trials in October 2000 for the treat-
ment of stage Ngastric cancer, colorectal can-
cer, and late-stage pancreatic cancer. These
trials have now been extended to include gas-
triclesophageal cancer, colorectal cancer, pan-
creatic cancer, and primary liver cancer.
11 SUMMARY
The involvement of more than one endocrine
hormone and neurotransmitter in the control
of gastric acid secretion has resulted in a num-
ber of therapeutic approaches directed toward
achieving its inhibition. Of these strategies
both muscarinic and histamine H2-receptor
antagonists have been used, with the latter
being the method of choice for the control of
acid-related disorders, particularly peptic ul-
cer disease, until the late 1980s. Along with
the successful treatment of many patients
Table 3.7 CCKJGastrin Receptor Antagonists That Have Been Evaluated in the Clinic
--- -

Woligand
Compound Identifier Company Binding (nM) Clinical Evaluation
Merck 8.5 Inhibition of gastric acid secretion: 50 mg, no
effect on basal acid secretion; 2.5,10,50
mg p.0. inhibited pentagastrin-stimulated
acid secretion (0.05-2 pg kg-' h-') (227).
Inhibition of CCK-4 induced panic attacks:
50 mg p.0. reduced frequency and intensity
of attacks (228); 30 mg p.0.o.d. for 7 days
failed to inhibit panic in patients (229).

Rotta Inhibition of gastric acid secretion: CR2194

(1,2.5, or 7.5 mg kg-' h-l) dose-
dependently inhibited gastrin (6.4-800
pmol kg-' h-')-stimulated acid secretion.
CR2194 (7.5 mg kg-' h-l) inhibited basal
and sham fed acid secretion (230).

Parke Davis Inhibition of panic attacks: In a randomized,

double-blind study CI-988 (100 mg TID for
6 weeks) was compared to placebo in
patients with panic disorder.
All patients improved during treatment and
no difference in the weekly rate of panic
attacks was seen between the treatment
groups (231).
JB95008 James Black Foundation 10 Inhibition of gastric acid secretion: JB95008
(0.02and 0.4 m g kg-' h-l) produced 27
and 63%inhibition, respectively, of
pentagastrin (1 pg kg-' h-l).

N
H
0
NH

Inhibition of gastric acid secretion: YF476 (5,

10, and 25 mg p.0. 0.d. for 14 days) acutely
(day 1) inhibited inhibited meal-stimulated
acid secretion but tachyphylaxis was
produced after 14 days' administration
(232).

with H,-receptor antagonists, certain limita-
tions on efficacy became apparent, the most
significant of which were a lack of effective-
ness in a subset of patients during nocturnal
periods and that acid-rebound occurred upon
cessation of treatment. Both these limitations
were ascribed to the upregulation of other hor-
monal mechanisms during periods of elevated
pH. The realization that histamine and acetyl-
choline ultimately exerted their effect through
the K+/Ht-ATPase enzyme or proton pump
on the parietal cell, provided a potentially
more complete means of controlling acid se-
cretion and a new focus for medicinal chemis-
try. The currently available compounds that
control acid-related disorders by inhibiting
this enzyme do so in an irreversible manner,
achieving sustained acid inhibition.
PPIs and Hz-receptor antagonists between
them have successfully treated disorders
caused by increased gastric acid secretion in a
vast number of patients worldwide. Their use
has continued to be applicable even with the
identification of H. pylori, a bacterium that is
highly correlated with the incidence of ulcer
disease. Hz-receptor antagonists and PPIs
have proven to be highly effective, are well
tolerated, and are considered safe. Although
significant progress in obtaining reversible
K+/H'-ATPase inhibitors as well as C C W
gastrin-receptor antagonists has been made in
recent years, compounds from either of these
groups seem unlikely to dislodge the irrevers-
ible PPIs as the preferred treatment in acid-
related disorders at present. Rather, single-
enantiomer versions of compounds such as
omeprazole, because of their associated phar-
macokinetic advantage over the racemic com-
pounds, seem set to retain their position
among the highest earning prescription drugs
for some time to come.
12 ACKNOWLEDGMENTS
The authors thank Barret Kalindjian for help-
ful advice and encouragement during the com-
pletion of this chapter and Veronique Moris-
son for conducting literature searches on
certain compounds contained therein.
Inhibitors of Gastric Acid Secretion
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Cytokine

Contents
1 Introduction, 130
2 Chemokines and Chemokine Receptor Biology, 131
3 Chemokine Receptor Signaling, 134
4 Chemokine Receptor Structure, 135
5 Chemokine Ligand Structure, 135
6 CC Receptors, 138
6.1 CCR1, 138
6.1.1 CCRl Receptor Structure, 139
6.1.2 CCRl Antibodies, 139
6.1.3 CCRl Peptide Antagonists, 139
6.1.4 CCRl Small Molecule Antagonists, 139
6.2 CCR2, 143
6.2.1 CCR2 Receptor Structure, 143
6.2.2 CCR2 Antibodies, 144
6.2.3 CCR2 Peptide Antagonists, 144
6.2.4 CCR2 Small Molecule Antagonists, 145
6.3 CCR3, 148
6.3.1 CCR3 Receptor Structure, 149
6.3.2 CCR3 Antibodies, 149
6.3.3 CCR3 Peptide Antagonists, 149
6.3.4 CCR3 Small Molecule Antagonists, 150
6.4 CCR4, 152
6.5 CCR5,153
6.5.1 CCR5 Receptor Structure, 154
6.5.2 CCR5 Antibodies, 154
6.5.3 CCR5 Peptide Antagonists, 155
6.5.4 CCR5 Small Molecule Antagonists, 155
6.6 CCR6, 159
6.7 CCR7, 160
6.8 CCRS, 161
7 CXC Receptors, 161
7.1 CXCRl and CXCR2,161
7.1.1 CXCRl/CXCR2 Receptor Structure, 162
7.1.2 CXCRllCXCR2 Antibodies, 162
7.1.3 CXCRllCXCR2 Peptide Antagonists, 162

7.1.4 CXCRllCXCR2 Small Molecule
Antagonists, 163
7.2 CXCR3, 165
7.2.1 CXCR3 Antibodies, 166
7.2.2 CXCR3 Small Molecule Antagonists, 166
7.3 CXCR4 166
7.3.1 CXCR4 Receptor Structure, 166
7.3.2 CXCR4 Antibodies, 167
7.3.3 CXCR4 Peptide Antagonists, 167
7.3.4 CXCFi4 Small Molecule Antagonists, 168
8 Viral Chemokines and Chemokine Receptors, 170
9 Cytokines, 172
9.1 IL-1, 173
9.1.1 IL-1 Knockout and Transgenic Mice,
173
9.1.2 IL-1 ModulatorsIClinical Data, 173
9.2 IL-2, 174 - 9.8 TNFa, 182
9.2.1 IL-2 Knockout and Transgenic Mice,
175
9.2.2 IL-2 Modulators/Clinical Data, 175
9.3 IL-4, 175
9.3.1 IL-4 Knockout and Transgenic Mice,
176
9.3.2 IL-4 Modulators/Clinical Data, 176
1 INTRODUCTION
Cell/cell communication is an important event
in an organism's ability to respond to insult.
Cytokines and chemokines play an important
role in this process (1). Disease processes in
which one or more of these factors are overex-
pressed, or deficient, may benefit from modu-
lation of these cell-signaling events.
Cytokines can exist as both extracellular
soluble proteins (8-40 kDa) and cell surface
molecules. In response to insult, they are able
to regulate host defense, differentiation, cell
division, apoptosis, repair, and inflammation
in a variety of cell types. Originally termed
lymphokines and monokines, based on their
cellular source, it was eventually recognized
that these soluble mediators are more gener-
ally products of leukocytes. Because many of
them communicate signals between leuko-
cytes, many of the cytokines are now desig-
nated as interleukins. Cytokines are both
pleiotropic and redundant in their ability to
communicate and initiate biological re-
sponses. They can regulate cell function in an
autocrine, paracrine, or intercrine fashion
through cell surface receptors that are classi-
Chernokine and Cytokine Modulators
9.4 IL-5, 176
9.4.1 IL-5 Knockout and Transgenic Mice,
177
9.4.2 IL-5 Modulators/Clinical Data, 177
9.5 IL-6, 178
9.5.1 IL-6 Knockout and Transgenic Mice,
179
9.5.2 IL-6 Modulators/Clinical Data, 179
9.6 IL-12, 180
9.6.1 IL-12 Knockout and Transgenic Mice,
180
9.6.2 IL-12 ModulatorslClinical Data, 180
9.7 IL-13, 181
9.7.1 IL-13 Knockout and Transgenic Mice,
181
9.7.2 IL-13 ModulatorsIClinical Data, 181
9.8.1 TNFa Knockout and Transgenic Mice,
182
9.8.2 TNFa Modulators/Clinical Data, 182
9.9 IFNy, 183
9.9.1 IFNy Knockout and Transgenic Mice,
183
9.9.2 INFy Modulators/Clinical Data, 183
fied into families on the basis of structure and
function (2). Cytokine receptors are composed
of single-spanning membrane domains that
form high affinity complexes upon binding
with their ligands. Signaling does not require
high receptor occupancy and can occur with
very low concentrations of ligand, proceeding
through tyrosine phosphorylation using non-
receptor tyrosine kinases such as Janus kinase
(JAK). This cytoplasmic signal then triggers a
distinct cellular response. Recombinant pro-
teins have been evaluated therapeutically in
cytokine-deficient patients in an effort to en-
hance a desired physiologic function. For ex-
ample, erythropoietin has been used in cancer
patients on chemotherapy or in chronic renal
failure patients, as a method to stimulate red
blood cell production. In contrast, overproduc-
tion of cytokines may result in an untoward
response such as inflammation, cancer, or au-
toimmune disease, and blocking the produc-
tion or activity of these cytokines has also been
shown to have therapeutic benefit.
Chemokines and chemokine receptors play
an important role in chemotaxis (cell traffick-
ing). The term chemokine is derived from che-
motactic cytokine. Chemokines are protein
 Ihemokines and Chemokine Receptor Biology

Leukocyte

Extravasation

Endothelium

I
Heparan sulfate
I) Chemokine
"r Adhesion molecule
66 lntegrin
Chernokine receptor

Figure 4.1. Schematic representation of leukocyte transmigration induced by chemokines. The first
step involves rolling attributed to interaction between the leukocyte and the endothelial cells. This
process is mediated by selectins and selectin ligands expressed on the surface of both cell types. In the
next step a chemokine interacts with its receptor, inducing leukocyte activation and conformational
changes in the adhesion molecules (integrins) and resulting in firm adhesion to the endothelial
surface. It is believed that the chemokine is immobilized on the endothelial surface by interactions
with glycosaminoglycans. The leukocytes then transmigrate into the tissues.

lig,ands(8-10 kDa) that signal through inter-
adtion with seven-transmembrane G-protein-
CO'upled receptors (GPCRs). Two primary
nctions for chemokines are recruitment of
l e~kocytes
~ from circulation to a site of injury
or inflammation, and regulation of homeo-
st: itic leukocyte trafficking. In addition to be-
in1: able to initiate a specific cell migration,
chemokines are also implicated in leukocyte
ac.tivation, proliferation, angiogenesis, and
1~nphopoiesis, and can act as coreceptors for
HIW.Although the chemokine field has a
YOung history, significant advances have been
m:ide and modulators of chemokine/chemo-
kine receptor interaction are currently being
evaluated in clinical trials.
2 CHEMOKINES A N D C H E M O K I N E
RECEPTOR B I O L O G Y
Leukocyte migration to tissues is essential for
development of an inflammatory response.
The process involves two steps: arrest and
firm adhesion of circulating cells on endothe-
lial surfaces and migration through the endo-
thelium into the interstitium (Fig. 4.1) (3). It
has become clear in recent years that both pro-

*- Charged
-
'6% Uncharged
Figure 4.2. Schematic diagram of a two-dimensional model of a chemokine receptor. The extracel-
lular loops (ECL), the putative helical regions that traverse the cell membrane, and the intracellular
loops (ICL) are shown. The GPCR (family A) signature
- disulfide bridge between Cys residues of ECLl
and ECL2 is also shown.
cesses are regulated largely by a group of low
molecular weight cytokines, the chemokine
family. Over 40 chemokines have been identi-
fied to date (for reviews, see Refs. 4-7). Based
on the spacing of two amino terminal cys-
teines, they are classified into two major and
two minor families. In the CXC family ( afam-
ily) one amino acid separates the first two cys-
teines, whereas in the CC family (P family) the
two first cysteines are adjacent to each other.
The two minor families of chemokines include
only one member each, the C family repre-
sented by lymphotactin and the CX,C family
represented by fractalkine.
Chemokines exert their action through
seven-transmembrane receptors, which are
GPCRs (Fig. 4.2). Six CXC receptors, 10 CC
receptors, and one receptor each for lympho-
tactin and fractalkine have been identified.
Table 4.1 summarizes the human chemokine
receptor family, their ligand specificity, and
the cell types that predominantly express
these receptors. The new nomenclature for
chemokines is also included in the table (8).
Chemokine and Cytokine Modulators
Extracellular
One i m ~ o r t a n tfeature of the chemokine
and chemokine receptor family is the high de-
gree of promiscuity with regard to ligand bind-
ing. Only a few receptors have been identified
that bind with high affinity to only one ligand,
and most ligands interact with more than one
receptor. This apparent redundancy could be a
potential problem in the development of re-
ceptor antagonists as therapeutics. However,
it is not clear whether the overlapping activi-
ties of chemokines are relevant in the in uivo
situation. It may be that each chemokine plays
a specific function in a specific setting at a spe-
cific time, orchestrating a coordinated chemo-
tactic response. In addition, CXC chemokines
bind with high affinity only to CXC receptors
and CC chemokines bind to CC receptors, so
there is some degree of selectivity in the
family.
From the functional point of view, chemo-
kines from the CC family in general do not act
on neutrophils but attract monocytes, eosino-
phils, basophils, lymphocytes, and dendritic
cells (9,lO). (One notable exception is the abil-
 Table 4.1 Chemokine Receptor Familiesa
Name Cell typeb Ligandsc
- - - - - - -- - --

CXCRl Neutrophils IL-8 (CXCL8), GCP-2 (CXCLG)

CXCR2 Neutrophils IL-8 (CXCLS), GROa (CXCLl), GROP (CXCL2), GROy (CXCL3),
NAP4 (CXCL7), ENA-78 (CXCLS), GCP-2 (CXCL6)
cxcR3 T-cells (Thl and Tcl) IP-10 (CXCLlO),MIG (CXCLS), ITAC (CXCL11)
CXCRA Ndive T-cells, B-cells SDF-1 (CXCL12)
cxcR.5 B-cells, subset of T-cells BCA-11BLC (CXCL13)
CXCR6 T h l and Tcl cells CXCL16
CCRl Activated T-cells, monocytes, eosinophils, immature MIP-la (CCL31, MIP-1P (CCL4), MCP-3 (CCL7), RANTES (CCL5)
dendritic cells, basophils
Monocytes, macrophages, activated T-cells, NK cells MCP-1 (CCL2), 2 (CCL8), 3 (CCL7), 4(CCL13)
Eosinophils, basophils, activated T-cells (Th2) Eotaxin (CCLll), eotaxin 2 (CCL24), eotaxin 3 (CCL261, MCP-3
(CCL7), MCP-4 (CCL13), RANTES (CCL5)
d
W
CCRA Activated T-cells, basophils, platelets TARC (CCL17), MDC (CCL22)
W CCR5 Activated T-cells, monocyte/macrophages, immature MIP-1P (CCL4), RANTES (CCL5), MIP-la (CCL3)
dendritic cells
CCR6 T-cells, immature dendritic cells, B-cells MIP-3(w/LARC/exodus (CCL20)
CCR7 Mature dendritic cells, subset T- and B-cells MIP-3pIELC (CCLlg), SLC/6Ckine/exodus 2 (CCL21)
CCRB Monocyte/macrophages, activated T-cells (Th2) 1-309 (CCL1)
CCR9 Thymocytes, T-cells TECK (CCL25)
CCRlO T-cells, Langerhans cells CTAWEskine (CCL27), MEClCCKl (CCL28)
CX,CRl T-cells, natural killer cells Fractalkine/newotactin (CX,CLl)
XCRl T-cells LvmphotactidSCM-la (XCLl), SCM-1B (XCL2)
"Data from Refs. 9, 11, and 12.
*Thl, T helper cell type 1; Th2, T helper cell type2; Tcl, T cytotoxic cell type 1; Tc2, T cytotoxic cell type 2.
"BCA, B-cell-activating chemokine; BLC, B-lymphocyte chemoattractant; ELC, Epstein-Barr virus-induced receptor ligand chemokine; ENA-78, epithelial cell-derived neutro-
phil-activating factor, 78 amino acids; GCP-2, granulocyte chemoattradant protein; GRO, growth-related oncogene; IP-10, interferon-inducible protein 10; I-TAC, interferon-
inducible T-cell a chemoattractant; LARC, liver- and activation-related chemokine; MDC, macrophage-derivedchemokine; MIG, monokine induced by y interferon; MIP, monocyte
inflammatory protein; MCP, monocyte chemoattractant protein; NAP, neutrophil-activating protein; RANTES, regulated on activation, normal T expressed and secreted; SDF-1,
stromal cell-derived factor 1;SLC, secondary lymphoid tissue chemokine; T A X , thymus- and activation-related chemokine; TECK, thymus-expressed chemokine.

ity of CCRliMIP-la to generate a chemotactic
response for neutrophils in rodents.) On the
other hand, chemokines from the CXC family
can be further subdivided into two classes:
those that contain an ELR sequence, which
attract neutrophils, and the subset that lacks
the ELR motif, which attracts lymphocytes
(13, 14).
Based on their expression patterns, chemo-
kines can be classified as inducible or consti-
tutive. Inducible chemokines participate pri-
marily in inflammatory responses and are the
majority members of the family. The constitu-
tive chemokines are expressed primarily, but
not exclusively, in secondary lymphoid organs,
and recent evidence suggests they play a major
role in lymphocyte and dendritic cell homing
(15). It has been shown that these chemokines
can also be responsible for the organization of
specialized structures inside lymphoid organs,
such as formation of germinal centers (16).
Chemokine receptor expression is crucial for
determining the migration pattern of leuko-
cytes. Some receptors are restricted to certain
cell types. For example, CXCRl is expressed
predominantly in neutrophils, whereas others
like CCR2 are expressed on a variety of leuko-
cytes, including monocytes, T-cells, basophils,
dendritic cells, and natural killer cells. Most
leukocytes express more than one receptor at
any given time. Regulation of chemokine re-
ceptor expression has recently been shown to
occur upon activation or deactivation of mono-
cytes, dendritic cells, and T-cells (5, 15, 17).
3 CHEMOKINE RECEPTOR SIGNALING
The intracellular signals involved in chemo-
taxis are not yet fully understood, and much of
the information available today has been de-
duced from signaling information for other
GPCRs. However, significant new data re-
garding the chemotactic process have accumu-
lated in recent years. Like other seven-trans-
membrane receptors, chemokine receptors
couple to G-proteins. Many chemokine-in-
duced signaling events are inhibited by Borde-
tella pertussis toxin (PTX), suggesting that
chemokine receptors are linked to G-proteins
of the Gai class (18,19). In cotransfection ex-
periments it has been shown that CXCRl and
Chemokine and Cytokine Modulators
CXCR2 couple to Gai2, Gari3, Ga14, and Gal6
(20). Physical association between Gai and
several chemokine receptors has been docu-
mented (21,22). In some studies, PTX did not
completely block the calcium response, sug-
gesting the chemokine receptors may couple
to other G-proteins such as Gq or Ga16. In
addition, it has been suggested that the speci-
ficity of the coupling may be cell type specific
(23, 24).
Chemokine receptor activation leads to the
generation of a complex cascade of cellular
events, including the generation of inositol
triphosphates, the release of intracellular cal-
cium, and the activation of protein kinase C.
The release of the Goripy subunits from Gai
has been described as an essential step in this
process (25). However, the release from Gas or
Gaq does not result in induction of chemo-
taxis, suggesting that the py subunits are nec-
essary but not sufficient to induce cell migra-
tion and that Gai itself plays some role in the
process (26).
Activation of the chemokine receptor leads
to rapid activation of phosphoinositide-spe-
cific phospholipases, which leads to inositol-
1,4,5-triphosphate formation and a transient
rise in intracellular calcium (18). Phospho-
lipase C (PLC) isoforms that are involved in
chemokine receptor activation become acti-
vated by direct interaction with the by sub-
units. In addition to its interaction with PLCs,
the py subunits also interact with the type I,
phosphoinositol3 kinase y (PI3Ky), and acti-
vation of this enzyme results in the formation
of PtdIns(3,4,5)P3 (17). Mice that do not ex-
press PI3Ky have severely impaired chemo-
kine-stimulated signal transduction, and PKB
is not activated, suggesting an important role
for this pathway in the chemotactic process
(27-29). Although leukocytes isolated from
these mice showed a decrease in cell chemo-
taxis, the response is not completely lost, and
under conditions of complete PI3Ky inhibi-
tion, neutrophils can still chemotax in re-
sponse to chemokines (30).
Receptor dimerization upon ligand binding
has been described mostly for the growth fac-
tor receptor; however, recent reports have also
suggested heterodimerization for seven-trans-
membrane receptors, including chemokine re-
ceptors (31-33). It has been proposed that che-
Chemokine Ligand Structure
HOOC
igure 4.3. Schematic diagram showing the pro-
sed chemokine receptor signaling pathways. This
agram does not represent the complete pathway.
gand binding induces the exchange of GDP for
rP on the G protein and this causes dissociation of
e subunits. Gai decreases the levels of CAMPand
l y causes the activation of PLC and PI3Ky. PLC,
lospholipase C; DAG, diacylglycerol; IP3, inositol
iphosphate; PKC, protein kinase C; MAPK, mito-
n-activated protein kinase; PI3Ky, phosphoinosi-
13 kinase y; PKB, protein kinase B.
okine receptor dimerization results in the
!tivation of the Janus kinase-signal trans-
tcers and activator of transcription (JAK-
FAT) signaling pathway (34). Figure 4.3
ows a schematic of the pathways involved in
.emokinereceptor signaling.
CHEMOKlNE RECEPTOR STRUCTURE
had been speculated 20 years ago that
PCRs have a seven-transmembrane (TM)
helical motif that traverses the cell membrane
(Fig 4.3) (35-37). The recent elucidation of a
high resolution, three-dimensional (3D)X-ray
structure of rhodopsin has confirmed the
seven-transmembrane domain topology (38).
GPCRs are classified into four families (A-D),
with chemokine receptors belonging to the
type A (e.g., rhodopsin-like). These single-
chain, membrane-bound proteins have an ex-
tracellular N-terminal (NT) domain and three
extracellular loops (ECL1-ECL3) that act in
concert to bind a chemokine ligand (Fig. 4.2).
The cytoplasmic domain of the receptor is
composed of three loops and a C-terminal seg-
ment, which also act in concert to transduce
the chemokine signal. The core of the receptor
is defined by seven-transmembrane spanning
helices. Based on the rhodopsin X-ray struc-
ture, the TM-a helices vary in length and can
extend beyond the lipid bilayer. The orienta-
tion of the TMs impose a stereo and geometric
specificity on a ligand's ability to enter the TM
binding domain. The TM core is mainly
formed by transmembrane a-helices 2,3,5,6,
and 7. Chemokine receptors, like other A-fam-
ily GPCRs, have the conserved signature
amino acid sequence in all seven TMs (TM1
GN, TM2 D, TM3 DRY, TM4 P, TM5 P, TM6
W, and TM7 NP) (Figs. 4.4a, 4.4b, 4.4c, 4.4d,
and 4.5). There are also conserved amino acid
residues that are a unique signature of chemo-
kine receptors. These residues are the Cys-Cys
motif in TM7, an acidic Glu residue (E) in
TM7, and cysteines in both the N-termini and
ECL3 that form a disulfide bond (Fig. 4.2).
5 CHEMOKINE LICAND STRUCTURE
Remarkable progress has been made in solv-
ing the molecular structure of chemokines by
high resolution X-ray crystallography and
NMR spectroscopy (39-48). Regardless of the
low degree of sequence homology among the
chemokines, they all exhibit a common folding
pattern that adopts a three-stranded antipar-
allel P-sheet, to which a C-terminal short a-he-
lix is obliquely attached (Fig. 4.6). Intramolec-
ular disulfide bonds stabilize this structure.
The N-terminal amino acids preceding the
first conserved cysteine have a large degree of
movement, which renders structural studies

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NGKLLLAVFYCLLFVFSLLGNSLVILVLVV
FASHFLPPLYWLVFIVGALGNSLVILVYWY
FSRAFQPSVSLTVAALGLAGNGLVLATHLA
CXCR1-HUMAN LNKYVVIIAYALVFLLSLLGNSLVMLVILY
CXCRZ HUMAN INKYFWIIYALVFLLSLLGNSLVMLVILY
CXCR~IHUMAN FDRAFLPALYSLLFLLGLLGNGAVAAVLLS
CXCR4-HUMAN FNKIFLPTIYSIIFLTGIVGNGLVILVMGY
CXCR5-HUMAN FKAVNPVAYSLIFLLGVIGNVLVLVILER
DAMCKILSGFYYTGLYSEIFFIILLTIDRYLAI
NAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAI
HGMCKLLSGFYHTGLYSEIFFIILLTIDRYLAI
LGLCKMISWMYLVGFYSGIFFVMLMSIDRYLAI
NTMCQLLTGLYFIGFFSGIFFIILLTIDRYLAV
NATCKLLKGIYAINFNCGMLLLTCISMDRYIAI
VHFCKLIFAIYKMSFFSGMLLLLCISIDRYVAI
TVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAV
TFMCKVVNSMYKMNFYSCVLLIMCISWRYIAI
SATCRTISGLYSASFHAGFLFLACISADRYVAI
CXCR1-HUMAN TFLCKVVSLLKEVNFYSGILLLACISVDRYLAI
CXCR2-HUMAN TFLCINVSLLKEVNFYSGILLLACISVDRYLAI
CXCR3-HUMAN SGLCKVAGALFNINFYAGALLLACISFDRYLNI
CXCR4-HUMAN NFLCKAVHVIYTVNLYSSVLILAFISLDRYLAI
CXCRS-HUMAN TFLCKTVIALHKVNFYCSSLLLACIAVDRYLAI
CCR1-HUMAN KLFQALKLNLFGLVLPLLVMIICYTG
CCRZ HUMAN NNFHTIMRNILGLVLPLLIMVICYSG
CCR~IHUMAN RHFHTLRMTIFCLVLPLLVMAICYTG
CCR4-HUMAN KVLSSLEINILGLVIPLGIMLFCYSM
CCR5-HUMAN KNFQTLKIVILGLVLPLLVMVICYSG
CCR6-HUMAN KLLMLGLELLFGFFIPLMFMIFCYTF
CCR7-HUMAN FITIQVAQMVIGFLVPLLAMSFCYLV
CCR8-HUMAN KIFTNFKMNILGLLIPFTIFMFCYIK
CCR9-HUMAN KSAVLTLKVILGFFLPFWMACCYTI
CCRlO-HUMA KGASAVAQVALGFALPLGVMVACYAL
CXCRl-HUMAN RMVLRILPHTFGFIVPLFVMLFCYGF
CXCR2-HUMAN RMLLRILPQSFGFIVPLLIMLFCYGF
CXCR3-HUMAN RTALRVLQLVAGFLLPLLVMAYCYAH
CXCR4-HUMAN VVVFQFQHIMVGLILPGIVILSCYCI
CXCRS-HUMAN WFTSRFLYHVAGFLLPMLVMGWCYVG
Figure 4.4. Sequence alignment of human CC and CXC che-
mokine receptors. For clarity, only the predicted seven (I-VII)
transmembrane helical domains are shown.
Chemokine and Cytokine Modulators
HEL-2
CCR1-HUMAN MTSIYLLNLAISDLLFLFTLPFWIDYKLKD
CCR2-HUMAN LTDIYLLNLAISDLLFLITLPLWAHSAANE
CCR3-HUMAN MTNIYLLNLAISDLLFLVTLPFWIHWRGH
CCR4 HUMAN MTDWLLNLAISDLLFVFSLPFWGYYAADO-
MTDIYLLNLAISDLFFLLTVPFWAHYAAAQ
MTDWLLNMAIADILFVLTLPFWAVSHATG
MTDTYLLNLAVADILFLLTLPFWAYSAAKS
ITDVYLLNLALSDLLFVFSFPFQTYYLLDQ
MTDMFLLNLAIADLLFLVTLPFWAIAAADQ
PTSAHLLQLALADLLLALTLPFAAAGALQG
VTDWLLNLALADLLFALTLPIWAASKVNG
VTDWLLNLALADLLFALTLPIWAASKVNG
STDTFLLHLAVADTLLVLTLPLWAVDAAVQ
MTDKYRLHLSVADLLFVITLPFWAVDAVAN
STETFLFHLAVADLLLVFILPFAVAEGSVG
TFGVITSIIIWALAILASMPGLY
TFGWTSVITWLVAVFASVPGII
TFGVITSIVTWGLAVLAALPEFI
TYGVITSLATWSVAVFASLPGFL
PRSKIICLVWJGLSVIISSSTFV
LISKLSCVGIWILATVLSIPELL
RMGTTLCLAVWLTAIMATIPLLV
LYSKMVCFTIWVLAAALCIPEIL
GRAHLVSVIVWLLSLLLALPALL
CXCR1-HUMAN HLVKFVCLGCWGLSMNLSLPFFL
CXCR2-HUMAN YLVKFICLSIWGLSLLLALPVLL
CXCR3-HUMAN ARVTLTCLAVWGLCLLFALPDFI
CXCR4-HUMAN LAEKVVYVGVWIPALLLTIPDFI
CXCRS-HUMAN LSIHITCGTIWLVGFLLALPEIL
CCR1-HUMAN KSKAVRLIFVIMIIFFLFWTPYNLTILISVF
CCR2 HUMAN RHRAVRVIFTIMIVYFLFWTPYNIVILLNTF
KYKAIRLIFVIMAVFFIFWTPYNVAILLSSY
KNKAVKMIFAVWLFLGFWTPYNIVLFLETL
RHRAVRLIFTIMIVYFLFWAPYNIVLLLNTF
RHKAIRVIIAWLVFLACQIPHNMVLLVTAA
RNKAIKVIIAVVVVFIVFQLPYNGWLAQTV
KTKAIRLVLIWIASLLFWVPFNWLFLTSL
KHKALKVTITVLTVFVLSQFPYNCILLVQTI
RRRALRVWALVAAFWLQLPYSLALLLDTA
CXCR1-HUMAN KHRAMRVIFAWLIFLLCWLPYNLVLLADTL
CXCR2-HUMAN KHRAMRVIFAWLIFLLCWLPYNLVLLADTL
CXCR3 HUMAN RLRAMRLVVVVWAFALCWTPYHLWLVDIL
KRKALKTTVILILAFFACWLPYYIGISIDSF
RQKAVRVAILVTSIFFLCWSPYHIVIFLDTL
CCR1- HUMRN LAVQVTEVIAYTHCCVNPVIY
CCR2 HUMAN OATOVTETLGMTHCCINPIIY
C C R HUMAN
~ ~ ~VM~VTEVIAYSHCCMNPVIY
CCR4- HUMAN YAIQATETLAFVHCCLNPIIY
CCR5- HUMAN QAMQVTETLGMTHCCINPIIY
CCR6- HUMAN YTKTVTEVLAFLHCCLNPVLY
CCR7- HUMAN IAYDVTYSLACVRCCVNPFLY
CCR8- HUMAN YATHVTIIISFTHCCVNPVIY
CCR9- HUMAN ICFQVTQTIAFFHSCLNPVLY
CCRlO-HUMAN VALLVTSGLALARCGLNPVLY
CXCRl HUMAN RALDATEILGFLHSCLNPIIY
CXCR~-HUMAN RALDATEILGILHSCLNPLIY
C X C R 3 ~HUMAN VAKSVTSGLGYMHCCLNPLLY
CXCRB- HUMAN KWISIT~LAFFHCCLNPILY
CXCRS- HUMAN VAITMCEFLGLAHCCLNPMLY
5 Chemokine Ligand Structure
difficult. Although chemokines exhibit similar
tertiary structures, their quaternary struc-
tures are different. As revealed by X-ray crys-
tallography and NMR spectroscopy, they form
dimers and tetramers. However, the mono-
meric form is believed to be the major species
found at physiological concentrations and,
based on numerous studies, monomers have
been shown to be the functional ligand (40,
49-51). The general consensus of these stud-
ies is that much of the binding energy and
selectivity for chemokines with their receptors
comes from the core domain. In contrast the
flexible N-terminal domain is required for re-
ceptor activation and signaling.
Mutation and chimera studies of IL-8
(CXCL8) have identified the ELR motif in the
N-terminal domain of IL-8, MGSA (CXCLl),
and NAP-2 (CXCL7) as essential for activa-
tion of CXCRl and CXCR2 (13). Similar stud-
ies with SDF-1 revealed that Lysl and Pro2
2" c y s
C-terminal
Figure 4.6. Monomer structure of the monocyte
chemotactic protein 1 (MCP-1). The structure was
retrieved from the Brookhaven data bank (http:/I
www.pdb.bnl.gov/) and displayed by Insight soft-
ware (Molecular Simulations, San Diego, CA).
Figure 4.5. Top view of a chemo-
kine binding site model (a), showing
only the helical backbone of resi-
dues in transmembrane domains I-
VII; the side chain for the conserved
Glu in TM (VII) is shown. Top view
of a biogenic amine binding site
v model (b), showing only the helical
backbone of residues in transmem-
brane domains I-VII; the side chain
for the conserved Asp residue in TM
(111) is shown.
are required for activation of CXCR4 (52).
Furthermore, although the N-terminus of
RANTES (CCL5) contributes significantly to
CCR5 activation, it adds little to the receptor-
binding affinity (53). Considering that one can
modulate signaling without significantly in-
terfering with binding, Simmons and cowork-
ers made a chemically derivatized form of
RANTES (AOP-RANTES), which retains
nanomolar receptor-binding activity yet
serves as a CCR5 antagonist (54). In a similar
study it has been shown that deletion of eight
or nine residues at the N-terminal of MCP-1
(CCL2) reduced the level of CCR2 binding by
less than seven times, and yet this truncation
completely inhibits chemotactic response (55-
57).
For most chemokines much of the binding
energy comes from the core domain, and cer-
tain surface residues have been identified as
contributing significantly to their binding af-
finity. The critical residues in RANTES and
IL-8 are F12 and 110, respectively. Mutation of
these to alanine caused 5000-fold reductions
in affinity for CCR3lCCR5 (RANTES recep-
tors) and 100-fold reductions in affinity for
CXCRl (IL-8 receptor) (58). Of the surface-
exposed residues in MCP-1, Y13 contributes
the most to MCP-1 binding affinity (55). In
contrast, the corresponding binding contribu-
tion of this aromatic residue in eotaxin is
much less than the corresponding residue in
other CC chemokines (59). Thus, residues im-
mediately following the first two cysteines
may in general be a common recognition ele-
ment in chemokines (58).
Additional hydrophobic residues between
the second cysteine and a 3-10 helix in the
"N-loop" have also been shown to be impor-
tant for chemokine binding. The N-loop in

IL-8 and MGSA is important for receptor
binding and for determining the specificity
(60). The key residues in IL-8 include Y13,
F17, F21, and I22 (60, 61). In RANTES, two
hydrophobic residues (I15 and L19) also con-
tribute significantly to CCR5 binding, as evi-
denced by mutation of these residues to Ala,
causing a >5000-fold reduction in affinity
(53). For MCP-1, mutation of many of the N-
loop residues had little or no effect. Small
changes were observed for two basic residues,
R18 and K19. It can be concluded that the
N-loop may be a common recognition element
involved in chemokine-receptor interactions,
and the nature and distribution of key resi-
dues may impart specificity. In MCP-1 two
clusters of primarily basic residues (R24, K35,
K49, and Y13), separated by a 35-A hydropho-
bic groove, reduced the level of binding by 15-
to 100-fold. Together these data suggest a
model in which a large surface area of MCP-1
contacts its receptor (CCRZ),and the accumu-
lation of a number of weak interactions results
in the 60-pM affinity observed for the wild-
type (WT) protein. In contrast, an Ala scan of
eotaxin (CCR3 ligand) has shown that resi-
dues contributing to receptor-binding affinity
and those required for triggering receptor ac-
tivation are relatively diffuse and distributed
throughout the N-terminal and N-loop re-
gions (59). The receptor-binding sites for vari-
ety of chemokines cover both similar and yet
significantly different sites, and these studies
may provide insight into the issue of receptor
specificity.
6 CC RECEPTORS
6.1 CCRl
CCRl was the first isolated CC chemokine re-
ceptor. Originally identified as the RANTES
(CCL5)/MIP-la (CCL3) receptor (62, 631, it
was subsequently shown that MCP-3 (CCL7)
also binds with high affinity to CCRl (64).
CCRl is expressed on various leukocyte cell
types, including monocytes, T-cells, and eosin-
ophils (see Table 4.1). In mouse, but not hu-
man, CCRl is also expressed on neutrophils
(65). CCRl mRNA has also been detected in
human dendritic cells (66).
Chernokine and Cytokine Modulators
A significant increase in CCRl ligand con-
centration for both MIP-la and RANTES has
been found in several inflammatory diseases
and animal models of those diseases. For ex-
ample, increases in MIP-la have been found
in the synovial Auid of patients with arthritis
(67) and in the cerebrospinal fluid of patients
with relapsing multiple sclerosis (MS) (68). In
a mouse model of MS, the experimental aller-
gic encephalomyelitis model (EAE), several
chemokines are upregulated, including MIP-la
and RANTES (69). In this model, RANTES
and MIP-la are produced exclusively by infil-
trating leukocytes (70,711. Moreover, neutral-
ization of MIP-la significantly blocks the de-
velopment of the initial and the relapsing
paralytic disease (72, 73). In spite of this
strong evidence regarding the role of MIP-la
in this disease, it has been difficult to establish
which receptor(s) mediates the MIP-la re-
sponse in this pathology. As shown in Table
4.1, this chemokine can bind with high affinity
to both CCRl and CCR5.
CCR1-I- mice show a normal distribution
of leukocytes, although trafficking and prolif-
eration of myeloid precursors are altered (74).
These mice are protected from pulmonary in-
flammation secondary to pancreatitis. Evi-
dence involving CCRl in transplant rejection
has also been obtained using knockout mice,
and these mice show a prolonged survival af-
ter heterotropic heart allografts. Low doses of
cyclosporin A, which would not be sufficient to
prolong graft survival, produced permanent
engraftment in these mice (751, suggesting
that this receptor plays a critical role in the
development of rejection. This has been con-
firmed with small molecule antagonists of
CCRl (see below). Recently, Blease and col-
leagues reported that fewer goblet cells and
less subepithelial fibrosis were found in
CCR1-I- mice compared to that found in wild-
type mice in a model of chronic fungal allergic
disease. IL-4 and IL-13 levels were lower in
the knockout mice but similar changes in leu-
kocyte recruitment and airway hyperreactiv-
ity were found in the two groups. Although
there was no effect on inflammation, these
data suggest a role for CCRl in airway remod-
eling, an important feature of asthma (76).
 6.1.1 CCRl Receptor Structure. Chimera
studies of CCRl show that the N-terminus is
important for ligand binding and selectivity
among chemokines, whereas this domain has
no effect on the receptor activation process, as
measured by calcium flux or chemotaxis as-
says (77). Instead, the ECL-3 of CCRl is
shown to be important for receptor signaling.
These data are consistent with a multisite
model for chemokine-chemokine receptor in-
teraction, in which one or more subsites deter-
mine chemokine selectivity, but others are
needed for receptor activation (78).
6.1.2 CCRl Antibodies. Antibodies to CCRl
kine ligands such as MIP-la and
ES have been reported and, although
some promiscuity based on
eir involvement with other chemokine re-
ptors, their biological activity is presumed to
based on upregulation of
ease models. Antibodies to
sed in an EAE model and
e demonstrated involvement of the ligand
TES have been effec-
-induced arthritis
odel, decreasing severity of the ongoing dis-
matory and fibrotic changes (80).
6.1.3 CCRl Peptide Antagonists. Modified
Met-RANTES
corporates a methionine at the N-terminus
f the ligand and has been shown to be an
on binding studies with
(81). It selectively inhibits THP-1
ediated by RANTES (IC,, = 6
or MIP-la (IC,, = 0.5 nM), while showing
response to MCP-1-mediated chemotaxis
d calcium flux. Met-RANTES has also been
wn to be effective in a model of arthritis,
rease in onset and severity of
d arthritis (CIA) when admin-
duction (82). AOP-RANTES,
entane that is isosteric with
side chain of methionine, also functions as
CR1 antagonist. In a rat experimental glo-
merulonephritis model, AOP-RANTES effec-
tively blocks monocyte infiltration (83). Be-
cause RANTES is also a ligand for the CCR5
receptor, it may be that these modified ligands
exert efficacy through dual antagonism.
6.1.4 CCRl Small Molecule Antagonists.
RP23618 (1) is an early example of a nonpep-
tide small molecule chemokine antagonist
(84). This compound inhibits lZ5I-RANTES
binding to THP-1 cell membranes (IC,, = 3
piW. The chemokine receptors CCRl and
CCR2 have been detected by RT-PCR as being
present on this cell type (62). However, be-
cause (1)inhibits RANTES-induced chemo-
taxis and has no effect on an MCP-l-stimu-
lated chemotactic response, this compound
appears to be CCRl selective. Because (1)is
not competitive with RANTES, it is presumed
to bind at an allosteric site.
There is a limited structure-activity rela-
tionship (SAR) provided around this series.
However, an optimal ch& length of three car-
bon atoms between the phenothiazine and the
piperidine is demonstrated. The two-carbon-
linked homolog has significantly reduced ac-
tivity (IC,, < 20% at 30 a) and the four-
carbon tether is at least 3 times less active
than (1).
A similar pharmacophore finding was
noted for early leads from the Berlex group
(85). HEK293 cells containing the CCRl re-
ceptor were evaluated for binding with an-
other CCRl ligand, MIP-la. The three-car-
bon-tethered dibenzothiepine (2) was a potent

inhibitor of '',I-MIP-la binding (IC,, = 44
nM), whereas the two-atom-linked analog (3)
did not show appreciable inhibition (IC,, = 5
a). In a separate study, the structurally re-
lated xanthene carboxamide (4) was identified
(86) as a CCRl antagonist for its ability to
inhibit lZ5I-MIP-labinding to human CCRl
transfected in CHO cells (IC,, = 510 nM). Re-
placement of the hexyl group with benzyl(5)
led to a significant loss of activity (IC,, >
10,000 nM). It is interesting to compare (2)
versus (4) in terms of a similar linking space
between the aryl group and the tricyclic moi-
ety. The conformational shape imposed by the
amide and/or the position of the piperidine
unit may be important considerations in the
SAR of CCRl antagonists.
Chemokine and Cytokine Modulators
An initial strategy to optimize the drugabil-
ity of (2) focused on two fragments with poten-
tial liabilities. The 4-hydroxy phenyl piperi-
dine is also present in haloperidol and
implicated with neurotoxicity, and the tricy-
clic dibenzothiepine is a common feature of
CNS-active drugs. As indicated in Table 4.2,
removal of the 4'-hydroxy led to a complete
loss of potency against CCR1.
Diphenylmethane is a common tricyclic re-
placement and this strategy was implemented
successfully, leading to (11)(Ki = 52 + 5 nM,
 141

Table 4.2 Consequence of Replacing Hydroxyl on the 4-Phenylpiperidine Unit

in Dibenzothiepine Seriesa

R'
R R' K, wvnb
OH 4-F-Ph 44219
OH Ph 203t28
H Ph 19+5%at3pM
CN Ph O%at3pM
C(O)CH, Ph O%at3pM
'%rornRef. 85.
bK, values derived from competitive binding on CCRl with lZ5IMIP-la.

against the same receptor from different spe-

cies, HEK293 cells were transfected with mar-
moset, rabbit, and mouse CCRl (88). Al-
though (11)is efficient in displacement of lZ5I-
MIP-la from human, rabbit, and marmoset, it
is ineffective against mouse CCRl (Table 4.3).
Species variability is an issue for many chemo-
kine receptors and presents a challenge for
evaluation of possible human therapeutics in
relevant animal models.
Many of the published chemokine antago-
nists have a basic nitrogen as a common fea-
ture, and it is interesting to speculate how this
might serve as an important recognition ele-
ment. As noted earlier, the protonated nitro-
C1
Table 4.3 Species Selectivity for (11) in Its
Ability to Inhibit CCRl from Human,
Rabbit, Marmoset, and Mousea
1261-MIP-ladisplacement). Compound (11) K, of Inhibition of
s to be selective for CCR1, in that it has MIP-la Binding
by (11) (nMIb
CCR5, and CXCR4, or a select panel of bio- H~~~~ 2 2 0.2 41 2 6
genic amine receptors (87). There are no in Rabbit 43 t 17.8 245 t 60
of Marmoset 81 t 23 451 r+_ 191
e variable species similarity between human Mouse 32 t 3.5 >10,000
and mouse sequences. The homology between "From Ref. 88.
mouse/human CCRl is 79%. In an effort to bK, of inhibition of lZ5I-MIP-la to HEK293 cells ex-
compare response of a CCRl antagonist pressing CCRl receptors from various species.
142
Table 4.4 Activity of Quaternary Ammonium Salts versus Nonquaternized Compoundsa
- -
Compound
"From Ref. 85.
bKivalues are derived from competitive binding on CCRl with 1251-MIP-la.
gen might interact with the conserved Glu in
TM7 (Fig. 4.5). This hypothesis is given cre-
dence by the finding that quaternary ammo-
nium salts will show enhanced affinity with
the receptor, implying a possible ion pairing
with a carboxylate (Table 4.4).
A similar potency enhancement is seen for
the xanthene carboxamide quaternary salt
(16) (CCR1 IC,, = 0.9 nM), where the simple
nonquaternized parent is 150 times less active
(86). Compound (16) can exist as cis and trans
isomers and a stereochemical preference is in-
dicated in CCR1-binding studies (Table 4.5).
Additionally, these investigators were inter-
ested in identifying a compound that was ef-
fective against human and murine CCR1, and
therefore they screened against both recep-
tors. The minor isomer, presumed to be the cis
Chemokine and Cytokine Modulators
isomer, was 50 and 100 times less active for
the human and murine receptors, respec-
tively. Compound (16) is a functional antago-
nist of MIP-la-stimulated CaZ+flux in U937
cells (IC,, = 0.73 nM). It also shows modest
inhibition of CCR3 but is selective against a
panel of other chemokine receptors (89).
BX471 (17) is a potent CCRl antagonist
that has demonstrated efficacy in animal mod-
els of multiple sclerosis and transplant rejec-
tion. It has been determined to be a reversible
and surmountable antagonist that can effec-
tively displace the radioiodinated CCRl li-
gands l Z 5 I - W T E S (Ki = 2.8 nM), 1251-
Table 4.5 Species Selectivity for (16) in
Its Ability to Inhibit CCRl from
Human and Mousea
- -
Species Major Isomer Minor Isomer
-- -
Human CCRl 0.9 47
Murine CCRl 5.8 740
"Inhibitory activity against 1251-MIP-labinding to hu-
man and mouse CCR1.
 -la (Ki and 1251-MCP-3(K,
= 1 nM), = 5.5
(90). BX471 demonstrates functional an-
nism in its ability to block Ca2+ flux and
ibit CDllb upregulation in a whole blood
has been measured in
d rat (F < 20%). Even
gh activity against the rat CCRl is 100
es less potent than against human CCR1,
d despite modest bioavailability, the com-
nstrate efficacy in the
model when administered subcutane-
nally, in the rat het-
tropic heart transplant rejection model, in
ich animals are given subtherapeutic doses
CsA, BX471 was able to prolong survival
variants of the CCR2 receptor,
CCR2A and CCR2B, differing at their C-termi-
nal tails. These two isoforms are derived from
native splicing (92).
nd to be the predominant form
monocytes and seems to traffic more effi-
tly than CCR2A to the cell surface in
ly transfected cell lines (93). All four
onocyte chemotactic proteins (MCPs) are
ds of CCR2 (see Table 4.1).
on monocytes, T-
d immature dendritic cells.
eshly isolated B-cells also show surface ex-
ression of CCR2 (94).
MCP-1 is elevated in several inflammatory
asthma, rheumatoid ar-
herosclerosis, and
nfiltrates and their cyto-
products are characteristic features of in-
ed tissue in rheumatoid arthritis (RA).
e synovial cells of RA patients, and animal
odels of the disease, produce elevated levels
MCP-1 (95-99). Peptide antagonists and
MCP-1 as a contributor
sease pathology. CCR2-I- and MCP-I-/-
receptor and its ligand
a crucial role in the recruitment of mono-
e the precursors
. A significant re-
id deposition was found
both CCR2-I- and MCP-1-I- mice in mod-
of atherosclerosis. This reduction was ac-
panied by fewer macrophages in the ath-
sclerotic lesions of these mice (100, 101).
The relevance of these findings to human dis-
ease was suggested by the presence of MCP-1
in human atheromatous plaques (102).
In a mouse model of allergic lung inflam-
mation, MCP-1 mRNA was significantly in-
creased after antigen challenge and appeared
to correlate with lung macrophage accumula-
tion (103). Using the same model, it has re-
cently been shown that MCP-1 neutralization
drastically diminished bronchial hyperreac-
tivity to methacholine and reduced inflamma-
tory cell infiltration into the lung (104). How-
ever, in this type of model a clear discrepancy
has been found between CCFW- and MCP-1-I-
mice. CCR2-'- mice present an enhanced pul-
monary allergic response against Aspergillus
fumigatus (105) or no difference at all in the
response to repeated ovalbumin sensitization
and challenges (106). MCP-1-I- mice, by con-
trast, show a decrease in T-helper 2 (Th2) type
T-cell responses, including a decrease in IL-4
production (107). The mechanism that ex-
plains how CCR2/MCP-1affect T-cell differen-
tiation is not clear, although it seems that they
exert their effect at different levels.
As research in the area progresses, it ap-
pears that several other chemokine receptors,
in addition to CCR5 and CXCR4, can act as
coreceptors for the HIV virus (7). One of these
receptors is CCR2. A mutation in the CCR2
gene that leads to a conservative amino acid
substitution (V64I) has been linked to a de-
creased rate in the progression of the disease
but not with initial susceptibility to infection
(108). The mechanism responsible for this is
not clear.
6.2.1 CCR2 Receptor Structure. Chimera
studies have shown that the N-terminus,
ECL-1, and ECL-2 of the CCR2 receptor are
essential for MCP-1 binding and receptor-me-
diated signaling (77).A detailed analysis of the
amino acid residues of ECL-1 in CCR2 has
shown N104 and El05 as being essential for
high affinity agonist binding but not involved
in receptor activation. In contrast, the
charged amino acid residue HlOO does not
contribute to ligand binding but is vital for
receptor activation and initiation of trans-
membrane signaling (109).
The role of acidic residues in TM7 and
ECL-2 of the CCR2 receptor has been evalu-

ated for their contribution to the binding af-
finity of MCP-1 and several small molecule an-
tagonists (110). In this study, glutamate 291
(Glu291 or E291), a highly conserved acidic
residue in TM7 of most chemokine receptors,
has been identified to be critical for binding of
certain small molecule antagonists, and to a
lesser extent the binding of MCP-1 to its re-
ceptor (Fig. 4.5). It is hypothesized that the
acidic moiety interacts with the piperidine ba-
sic nitrogen of small molecule antagonists
(18-20). Because Glu291 is also involved in
MCP-1 binding to CCR2, the spiropiperidine
blockade of MCP-1 binding occurs by occupa-
tion of some of the space that CCR2-bound
MCP-1 occupies. A class of antagonists that
does not have a basic nitrogen (e.g., 27) is not
affected by mutation of Glu291, suggesting a
different binding motif for this series.
The acidic residue (Glu) in TM7 is found in
most chemokine receptors and is rare in other
serpentine receptors (Figs. 4.4 and 4.5). This
negatively charged residue may also be signif-
icant in the small molecule ligand binding of
other chemokine receptors. It is interesting to
note that receptor models indicate that
Glu291 is inside the ovid-helical bundle (Fig.
4.5) in a mirror image position to the critical
binding residue of biogenic arnine receptors,
an aspartic acid in TM3. This observation
might explain the fact that many of the leads
from high throughput screening for the che-
mokine receptors are compounds originally
synthesized for biogenic amine programs.
6.2.2 CCR2 Antibodies. A CCR2 monoclo-
nal antibody (mAb), ID9, was shown to have
therapeutic benefit in a primate model of re-
stenosis (111).The inflammatory response af-
ter stent implantation involves sustained ac-
cumulation of macrophages, resulting in
neointimal hyperplasia. The CCR2 mAb ID9
reduced intimal thickening in the area of the
stent approximately 60%, as ascertained on
day 28. Two CCR2-specific monoclonal anti-
bodies, MCP-1R02 and MCP-1R05, are re-
ported to antagonize MCP-1 activity on CCR2-
expressing cell lines and normal human cells
(112).These antibodies have been used to map
the HIV-1 binding site on CCR2. MCP-1R02 is
claimed to be specific for the amino terminus
and acts as an agonist; it suppresses HIV-1
Chemokine and Cytokine Modulators
replication in M- and T-trophic HIV-1 viral
isolates. MCP-1R05 is specific for ECL-3 of
CCR2, acts as an antagonist (31), and has no
effect on HIV-1 replication. Therefore, the
HIV-1 interaction is primarily through the
amino terminus.
Neutralizing antibodies to the CCR2 ligand
MCP-1 have helped to validate its role in dis-
ease pathogenesis of arthritis and glomeru-
loritis. An antibody against rat MCP-1 has
been evaluated in a collagen-induced arthritis
model, and was shown to reduce joint swelling
of the ankle, and suppresses macrophage infil-
tration to the synovial tissue (113). In an ani-
mal model of impaired renal function, in
which MCP-1 was detected in glomeruli, vas-
cular endothelial cells, and tubular epithelial
cells of the injured kidney, anti MCP-1 mAbs
reduced glomerulosclerosis and improved re-
nal function (114). Anti-MCP-1 monoclonal
antibodies have also shown therapeutic bene-
fit in a kidney fibrosis model, through block-
ade of renal cell proliferation (115), and in a
delayed-type hypersensitivity (DTH) model in
which T-cell trafficking was inhibited (116).
Finally, as part of an effort to define the role of
a variety of chemokines in allergic inflamma-
tion and airway hyperresponsiveness, anti-
bodies to RANTES, MIP-la, MCP-1, and the
mouse-specific chemokine MCP-5 were evalu-
ated in a mouse model of asthma (117). In re-
sponse to an ovalbumin (OVA) challenge, the
neutralizing antibody to MCP-1 reduced bron-
chial hyperresponsiveness and blocked leuko-
cyte infiltration into the airways.
6.2.3 CCR2 Peptide Antagonists. Trunca-
tion or N-terminal deletion of select amino ac-
ids in MCP-1 results in a protein antagonist
that blocks Ca2+ flux and chemotactic re-
sponse (118,119). Truncation of the first nine
amino acids (MCP-1'10-761), or deletion of
amino acids 2-8 (MCP-lC19-761, generate
+
functional CCR2 antagonists. These protein
antagonists have also demonstrated efficacy
in vivo. When MCP-1[9-761is administered
daily in the MRL-lpr arthritic mouse model,
there is a significant reduction in the onset of
arthritis, as determined by histopathologic
evaluation of the joint and reduced joint swell-
ing (57). Even when administered 12 days af-
ter disease onset, MCP-1[9-761 shows a marked
6 CC Receptors 145

Table 4.6 Selectivity Profile for CCR2 Antagonists (Spiropiperidines)versus Alpha

and S-HT Receptors

- - - - - - - - - - - - - -

Compound CCR2" Alpha la Alpha Id 5HTla

"Measured in THP-1 cells using 12'MCP-1.

reduction in symptoms and histopathology.

This suggests a turnover of cell infiltrate in
the lesion and indicates the potential for ther-
apeutic benefit through this mechanism after
disease onset. It is worthwhile to note that
ligand truncations have the potential to alter
receptor selectivity. For example, truncation
ofthe NH,-terminal amino acid in MCP-1 gen-
erates a strong activator of eosinophil chemo-
taxis (120), presumably acting on CCR3. Fur-
ther truncation converts the ligand to a CCR2
antagonist with no activity on eosinophils. Al-
though ligand truncations are a common ma-
nipulation to profile chemokine SAR, few
studies screen for selectivity of these trun-
cated ligands against other receptors.

6.2.4 CCR2 Small Molecule Antagonists.

The in vivo efficacy seen with anti-MCP-1an-
tibodies and peptide antagonists prompts the
search for small molecule CCR2 antagonists.
The spiropiperidines (18-20) are early exam-
ples of CCR2 antagonists, as determined by
'251-MCP-1binding and chemotaxis in THP-1
cells (110). As noted above with the CCRl an-
tagonists, a basic nitrogen is a common fea-
ture within this class of antagonists. Site-di-
reded mutagenesis suggests that this nitrogen
may form a critical recognition with an an-
ionic residue (E291)in helix 7. The orthogonal
relationship imposed on the phenyurethane
and the piperidine ring by the spiro-carbon is
urported to be essential for activity.
Selectivity is an issue for the spiropiperi-
dines, given that this class of compounds potent CCR2 antagonist still retained signifi-
shows appreciable activity against alpha ad- cant Alpha l a adrenergic activity (Table 4.6).
renergic and 5-hydroxytryptamine (5-HT) re- Another example of GPCR selectivity is-
ceptors. Although lead optimization improved sues for CCR2 antagonists is seen with the
CCR2 affinity (18)versus (20), and improved indolopiperidines (121) (Table 4.7). As might
the relative difference in selectivity, the most be anticipated by the initial lead (21), prob-
146
Table 4.7 Selectivity Profile of CCR2B Antagonists (Indolopiperidenes)versus 5-HT
and Dopaminergic Receptors
lematic affinity with 5-HT and dopaminergic
receptors was evident. However, impressive
improvements in selectivity were effected by
increasing the steric bulk around the piperi-
dine nitrogen, leading to a conformationally
rigid ring system (122) in the form of indo-
lotropane (22),or imposing conformational re-
striction in the linker as with the cis-cyclo-
hexyl derivative (23). C-2 substituents on the
Chemokine and Cytokine Modulators
pKi
indole ring were not well tolerated, presum-
ably because of the unfavorable influence on
the piperidine ring conformation. Incorpora-
tion of both the tropane and cyclohexyl moi-
eties simultaneously had a dramatic additive
effect in providing a potent CCR2 antagonist
(24) with good selectivity profiles.
TAK-779 (25) is recognized primarily as a
potent CCR5 antagonist, but it does exhibit
 CC Receptors

P~~~~
C1

g + ) OH \ I \
N
H

(23)

modest affinity for CCR2 (123). This is not
surprising, considering the close homology be-
tween the CCR2 and CCR5 receptors (71%
amino acid identity). The binding of lZ5I-
MCP-1 to a CHO cell line containing CCR2b
was inhibited by TAK-779 (IC,, = 27 nM),
which showed no effect against CCR1, CCR3,
CCR4, and CXCR4.
There have been no reports on in vivo eval-
uation of small molecule CCR2 antagonists.

P~~~~
C1

H \,,\\\"' o ~

\ I \
N
H

(24)

(25)
148
This may be a consequence of dissimilar re-
sponses against the human and murine recep-
tors, with these antagonists being less potent
against mouse and rat CCR2. This activity dis-
crepancy is noted in the case of (26),in which
significant potency is demonstrated against
the human receptor (binding IC,, = 54 nM,
chemotaxis IC,, = 16 nM), and yet it is ap-
proximately 100 times less potent against the
murine chemotactic response (IC,, = 1400
nM) (124).
Nonbasic CCR2 antagonists have also been
reported. The 3-chlorobenzyl 2-pyrrole car-
boxylic acid (27) is a modest CCR2 antagonist
(IC,, = 11.9 p N ) , and binding does not appear
to be influenced by mutation of the conserved
glutamic acid in TM7 (110). Consequently, it
may bind in a region distinct from the antag-
onists that contain a basic nitrogen. The
closely related indole (28)is also reported to be
a modest CCRZ antagonist (ICE+ = 1.2 a (CCL241,itsd eotaxin 3
(125).
Chemokine and Cytokine Modulators
6.3 CCR3
CCR3 was originally reported to be ex-
pressed on eosinophils and appears to be the
primary chemokine receptor on these cells
(126). It is also expressed on basophils, Th2
lymphocytes, and mast cells (127-129).
Based on its presence in a large array of cell
types that are crucial for the induction and
maintenance of inflammation in asthma and
other allergic diseases, there is a significant
effort to discover CCR3 receptor antago-
nists. In 1993, eotaxin (CCL11) was identi-
fied as an eosinophil-specific CC chemokine
(130, 131). Subsequently, CCR3 was found
to be activated by several other CC chemo-
kines including MCP-3 (CCL7), MCP-4
(CCL13), RANTES (CCL51, eotaxin 2
(CCL26) (126{ 1 3 g I
133).
 In human asthma, increased mRNA and
protein expression of eotaxin have been dem-
onstrated, as well as CCR3 expression on local
infiltrating eosinophils, as determined by bi-
opsies (134). Results obtained from mice ge-
netically deficient for the eotaxin gene (1351,
as well as studies using eotaxin-blockinganti-
bodies (117), have demonstrated a significant
effect on the course of a lung allergic reaction.
By contrast, another report describing the re-
sponse of eotaxin-deficient mice, in a similar
model of allergic lung inflammation, showed
no significant difference from that of wild-type
(136). The reason for this discrepancy is not
understood. CCR3-/- mice have shown that
this receptor plays an important role in eo-
nophil migration into the lung and skin. In
traperitoneally (i.p.) sensitized CCR3-/-
mice, eosinophil recruitment to bronchoalveo-
lar lavage fluid and lung is decreased com-
pared to that of wild-type after aerosol chal-
lenge. However, airway hyperreactivity
(AHR) is enhanced under the same conditions
(137). If the mice are sensitized epicutane-
ously with the same antigen, a diminished
AHR response and eosinophil recruitment is
observed (138). In the i.p. sensitized mice
here is a significant mobilization of mast cells
to the airway epithelium, but not into the
kin. These results suggest that the mecha-
m of induction of AHR might be different,
ending on the route of sensitization.
6.3.1 CCR3 Receptor Structure. Chimera
dies of CCR3 and CCRl receptors show
at the N-terminal segments of CCR3 appear
be important for eotaxin binding (77). How-
ver, eotaxin remained an effective agonist at
his chimeric receptor in either calcium flux or
emotaxis assays. These data are consistent
th a multisite model for chemokine-chemo-
ne receptor interaction.
6.3.2 CCR3 Antibodies. A specific CCR3
onoclonal antibody, 7Bl1, blocks binding of
taxin, MCP-3, and RANTES to the CCR3
ceptor, and also blocks chemokine-induced
emotaxis and calcium flux in human eosin-
phils (126). Because the CCR3 receptor is
resent on basophils, it is not surprising that
B11 blocks chemotaxis of these cells in re-
onse to CCR3 ligands (127). In these same
studies, 7 B l l was shown to inhibit histamine
and leukotriene release mediated by eotaxin.
This mAb has also been shown to inhibit the
release of reactive oxygen species (ROS) after
stimulation with eotaxin-1 or eotaxin-2 (1391,
and blocks the chemotactic response of eosin-
ophils on H W E C cells stimulated with TNFa
and IFNy (140). Because CCR3 has been im-
plicated in having a role in HIV-1 pathology,
7 B l l has also been used to define the involve-
ment of CCR3 in contributing to HIV-1-spe-
cific pathology (141).
CCR3 monoclonal antibodies have also
been evaluated in vivo. A rat mAb specific for
mouse CCR3 receptor depletes blood eosino-
phi1 levels in Nippostrongyos brasiliensis-in-
fected mice, and reduced eosinophil levels in
lung tissue and bronchoalveolar lavage fluid
(BAL) fluid after repeated treatment (142).
Surprisingly, this antibody had no effect on
CCR3-regulated Ca2+flux. In another report,
a guinea pig-specific CCR3 mAb, 2A8, was also
evaluated both in vitro and in vivo (143). 2A8
blocks the binding of guinea pig eotaxin to
guinea pig eosinophils and guinea pig CCR3
transfectants, and shows functional antago-
nism of guinea pig eosinophils, as measured by
Ca2+ flux and chemotaxis. In animals pre-
treated with lllIn-labeled eosinophils, 2A8 in-
hibited accumulation of these eosinophils in
response to eotaxin.
6.3.3 CCR3 Peptide Antagonists. As dem-
onstrated for other chemokines, modification
of the N-terminal region has been employed as
a successful strategy to identify receptor an-
tagonists. Truncation of MCP-3 (MCP3110-7611
provides a ligand that retains potent receptor
binding, yet is unable to induce chemotaxis,
enzyme release, or Ca2+flux (144). Dipeptidyl
peptidase is able to truncate RANTES'1-681to
RJW~~s[3-681 and eotaxin[l-741 toeo&[3-741 9
and both have demonstrated functional antago-
nism in vitro (145-147).
RANTES, which can signal through CCR1,
CCR3, or CCR5, has been modified to Met-
RANTES, as described earlier. Met-RANTES
acts as a functional antagonist and blocks hu-
man eosinophil chemotaxis, Ca2+ flux, actin
polymerization, and release of ROS after stim-
ulation with RANTES, MCP-3, and eotaxin
(148). Met-RANTES apparently antagonizes

the response of eosinophils through CCRl at
low concentrations and through CCR3 at
higher concentrations.
Met-chemokine P7 (Ckp7) is an N-termi-
nal-modified form of macrophage inflamma-
tory protein (MIP4), wherein an alanine is re-
placed with methionine at the extreme
N-terminal residue (149). MIP4 [also known
as pulmonary and activation-regulated che-
mokine (PARC) (150); alternative macro-
phage activation-associated CC chemokine
(AMAC) (151); or dendritic cell-derived che-
mokine (DCCK) (152, 15311 itself shows some
degree of CCR3 antagonistic activity. How-
ever, Met-Ckp7 is significantly more potent as
a CCR3 antagonist than MIP4, Met-RANTES,
or AOP-RANTES, and blocks eotaxin or
MCP4-induced eosinophil chemotaxis at con-
centrations as low as 1 nM. Direct binding of
CkP7 to CCR3 is evidenced by its ability to
displace radioiodinated eotaxin or MCP4 from
CCR3.
6.3.4 CCR3 Small Molecule Antagonists.
UCB 35625 (16),which has already been de-
scribed as having CCRl activity, also func-
tions as a CCR3 antagonist. This compound
inhibits a chemotactic response to transfected
cells expressing either CCRl or CCR3 (CCRlI
MIP-la IC,, = 9.6 nM, CCR3leotaxin IC,, =
93.7 nM) (89). Again, this dual activity is not
surprising, given the considerable homology
between the two receptors. Because (16) is not
as effective in ligand displacement binding as-
say as it is in blocking receptor function, this
suggests that it may bind with a common re-
gion in both CCRl and CCR3 that are neces-
sary for initiation of receptor signaling.
Another tool for measuring cellular re-
sponse is a shape-change assay, as measured
by gated autofluorescence1forward scatter
(GAFS) through flow cytometry (154, 155).
Eosinophil shape change in response to
eotaxin was effectively blocked by (16) (89).
In an effort to improve CCR3-selective an-
tagonists, carboxamide derivatives based on
(16) were synthesized (156). Given that it was
presumed that the ammonium salt was form-
ing an important electrostatic interaction
with a carboxylate in the receptor, an m i n e
moiety was maintained within the template. The
2-(bemothiazo1ethio)acetamide (29) showed po-
Chemokine and Cytokine Modulators
tency for CCR3 with improved selectivity over
CCRl (CCR3 IC,, = 750 nM, CCRl IC,, =
7100 nM). Further derivatization of the aro-
matic groups, believed to be important for
binding to hydrophobic pockets, led to (30)
(CCR3 IC,, = 2.3 nM, CCRl IC,, = 1900 nM).
CCR3 functional antagonism was demon-
strated by its ability to block Ca2+ flux and
chemotaxis of human peripheral blood eosin-
ophils stimulated with eotaxin (157).
Other CCR3-specific antagonists are char-
acterized by the piperidine ureas (31-33),and
the di-substituted pyrrolidine (34) (158).
Again, a basic nitrogen, capable of protonation
at physiologic pH, appears to be a common
feature. It is important to note that the qua-
ternary ammonium salt (32)demonstrates en-
hanced potency, relative to that of its parent
(31) in binding, chemotaxis, and a whole-blood
GAFS assay (Table 4.8) (159).
Indolinopiperidines such as (34) were iden-
tified through random screening as CCR3 an-
tagonists (IC,, = 0.7 pM) (160). An H-bond
acceptor at the 3-position of the aryl urea and
a six-atom chain linkage were identified as im-
portant structural requirements for CCR3-
binding affinity. The rigidity of these indolin-
opiperidines was relaxed by incorporation of a
4-benzylpiperidine in its place, giving (35)
(IC,, = 0.02 p,iVf). Substitution around the pi-
peridine further improved potency to the
nanomolar range, as evidenced by (36) (IC,, =
0.001 pM).
The substituted pyrrolidines (37) and (38)
have been reported to inhibit the binding of
eotaxin in CCR3-transfected HEK cells (IC,,
-1 nM) (161). These antagonists also inhibit
eotaxin-induced calcium signal and eotaxin-
induced eosinophilia. It is notable that these
relatively compact, low MW analogs are able
to provide exceptional binding affinity.
An exception to the requirement of a basic
nitrogen in the pharmacophore of a CCR3 an-
tagonist is noted by the phenyalanine deriva-
tives (39- 41). High throughput screening us-
ing a FLIPR format to measure intracellular
calcium changes identified (39) as an early
lead (CCR3 IC,, = 535 nM) (162). Enantio-
specificity appears to be critical for optimal
antagonist/receptor interaction as replace-
ment of the L-configuration of tyrosine with
the D-amino acid results in greater than 200-
fold lower affinity. Lead optimization to (40)
demonstrated potent binding affinity (CCR3
IC,, = 5 nM)and functional antagonism, as
measured by inhibition of eosinophil chemotaxis
by use of eosinophils from allergic donors (IC,,
= 15 nM). No in uiuo data are reported for these
compounds, presumably because of the hydro-
lytic lability of the ester. An ester replacement
strategy identified 41 with a CCR3 with an IC5,
value of 45 nM. It has yet to be shown whether
any of these small molecule CCR3 antagonists
are effective in an animal model.
152 Chemokine and Cytokine Modulators

Table 4.8 Inhibition of Eotaxin Binding, Eotaxin-Induced Transfectant,

and Eosinophil Chemotaxis
IC50 (rn
Human
CCR3 L1.2" CCR3 L I . ~ ~ Eosinophil
c
Compound Binding Chemotaxis Chemotaxis

"IC,, values derived from competitive binding of lZ5Ieotaxin to CCR3 L1.2 transfectant cells.
bIC50values derived from inhibition of eotaxin-induced chemotaxis of the L1.2 transfectant cell line.
"IC5, values derived from inhibition of eotaxin-induced chemotaxis of human eosinophils.

6.4 CCR4 linked to Th2 cell polarization (165-167) and

CCM was originally cloned by Power and col-
leagues from a basophilic cell line (163). RNA
for CCR4 is expressed in stimulated T-cells,
B-cells, monocytes, platelets, and, to a lesser
extent, in basophils (163,164). CCR4 has been
the expression of CCR4 was strongly UPregu-
lated upon activation through the T-cell recep-
tor or by other c~tokinessuch as transforming
growth factor P (TGF-P) (166, 167). Two high
affinity ligands have been described for CCR4,

MDC (CCL22)and TARC (CCL17) (see Table
4.1). The role of the mouse ligand analogs has
been studied in murine models of asthma. In
ovalbumin-sensitized and -challenged mice,
an anti-MDC antibody inhibits eosinophil re-
cruitment to the lungs and protects against
methacholine-induced bronchial hyperreac-
tivity (168). Additionally, an anti-TARC
antibody has been shown to attenuate an
OVA-induced inflammation and airway hyper-
responsiveness (169). In contrast to these re-
sults obtained from the neutralization of
CCR4 ligands, the receptor knockout does not
show any phenotype different from that of
wild-type mice in the same models of allergic
lung inflammation (170). In human tissues,
CCR4 is expressed in all IL-4-positive cells in
the bronchial mucosa of asthmatic patients
that have been challenged 24 h before tissues
were collected (171). In the same study, CCR4
ligands were found to be highly expressed by
the airway epithelium in the same patient
population. It has also been suggested that
CCR4 and its ligands are also involved in other
inflammatory diseases such as psoriasis and
atopic dermatitis (172-174). These last obser-
vations correlate with the fact that CCR4-pos-
itive T-cells include all skin-homing cells (cu-
taneous lymphocyte antigen-positive cells)
and other memory T-cells but not intestinal
memory or ndive T-cells (a4p7-positive T-cells)
(175). This suggests a-role for CCR4 in tissue-
specific homing of T-cells; however, the rele-
vance of this tissue-specific homing to inflam-
matory skin diseases is not yet clear.
This receptor, originally cloned as ChemR13,
is part of the family of CC chemokine recep-
tors binding RANTES (CCL5), MIP-la
(CCL3),and MIP-1P (CCL4) (176-178). CCR5
is expressed on monocytes and some CD4+
T-lymphocyte subsets and its activation leads
to chemotaxis of these cells (7).

CCR5 and CXCR4 represent the most im-
portant coreceptors for the H N -1 virus (179).
CCR5 is involved in a sequential process, in
which the viral gp120 interacts first with CD4,
and then associates with the chemokine recep-
tor to allow envelope fusion and viral entry.
Strong genetic evidence supports the impor-
tance of CCR5 in HIV pathogenesis. Individu-
als homozygous for the A32 deletion are defi-
cient in the surface expression of CCR5 and
constitute the majority of the exposed but un-
infected population (180-183). This mutation
is represented at a high frequency in several
human populations (184).
CCR5 is also expressed in T-lymphocytes
found in synovial tissues from rheumatoid ar-
thritis patients. Although CCR5 appears to be
preferentially expressed on T h l lymphocytes
(165,166), a recent clinical study proposed an
important role for this receptor in asthma, a
classic Th2 disease. This study suggests that
individuals carrying the A32 deletion allele are
at reduced risk of developing asthma (185).
Two subsequent studies, however, suggest
that there is no genetic evidence linking
asthma and atopy with CCR5 A32 polyrnor-
phism (186,187). Further studies in the asth-
matic population as well as the use of pharma-
cological tools would be needed to delineate
the importance of CCR5 activation in allergic
asthma.
Some correlation has been reported be-
tween the A32 mutation and graft rejection. In
21 patients homozygous for the mutation, a
longer graft survival was observed compared
to that for the control group. The results sug-
gest a role for this receptor in transplant re-
jection (188).
6.5.1 CCR5 Receptor Structure. Many mu-
tagenesis and chimera studies have shown
that the N-terminus and the ECL-2 of the
CCR5 receptor are involved in ligand binding
(189).It has also been shown that the ECL-2 of
CCR5 is the major determinant for chemokine
binding specificity, whereas the amino-termi-
nal domain plays a more significant role for
human immunodeficiency virus (HIV-1) and
virus coreceptor function (190). However, a
potential involvement for the ECL-2 of CCR5
in HN-1 viral infectivity has been elucidated
by d b studies that use mAb 2D7, in which
Chemokine and Cytokine Modulators
the epitope maps to a peptide derived from the
ECL-2 domain, and inhibits both infection and
gp120 binding (191). The overlapping binding
site of chemokines and gp120 on the CCR5
amino terminus and ECL-2, as well as the in-
volvement of these residues in the epitopes of
d b s , suggests that these regions are signifi-
cantly exposed at the receptor surface. More
recent studies have focused on the contribu-
tion of specific charged and aromatic resides
(D2, Y3, Y10, D11, E18, and K26) in the amino
terminus of CCR5 and their role in both che-
mokine and gp120 high affinity binding
(192-197).
The role of the transmembrane helical do-
mains of CCR5 receptor has also been evalu-
ated. It has been shown that a hydrophobic
residue in TM7, M287, is critical for both
MIP-la binding and receptor activation (Fig.
4.4). However, this mutant remains biologi-
cally active in the HN-1 coreceptor fusion as-
say (198). In another study it has been shown
that glycine residue (G163) close to the extra-
cellular domain in TM4, is critical for gp120
binding and infections by all tested R5 isolates
of HIV-1 (199). It is interesting to note that
chemokine binding can be independent of
HIV-1 coreceptor activity and different TM
domains can be involved in gp120 and chemo-
kine binding to the CCR5 receptor.
6.5.2 CCR5 Antibodies. CCR5 d b s have
been reported by a number of groups (191,
200-202) and have proved useful in defining
structural determinants important for natu-
ral ligand binding, chemokine activity and re-
ceptor signaling, gp120 binding to CCR5, and
epitopes important for controlling HIV-1 en-
try. The mAbs 3A9,2D7, and PA12 all inhibit
HIV-1 entry and fusion, yet display significant
differences in their ability to block chemokine
binding, calcium flux, and chemotaxis. The
3A9 mAb was shown to bind to the N-terminus
and is ineffective as an inhibitor of MIP-la,
MIP-lp, and RANTES binding, and does not
block MIP-lp-induced chemotaxis at concen-
trations up to 100 f l .However, 3A9 blocks
infection of PHA-activated PBMCs by M-tro-
phic strains (ID,, = 0.5-2.3 pg/mL) (200). The
murine mAb PA12, whose epitope also maps
to the CCR5 N-terminus, has no effect on
RANTES-induced calcium flux and is only a
6 CC Receptors
modest inhibitor of HIV-1 fusion and entry,
yet strongly inhibits gp120 binding (201). In
contrast, the 2D7 binding site is mapped to the
second extracellular loop of CCR5 and com-
pletely blocks chemokine binding, Env bind-
ing, and viral infection (191). MAb 45531 also
binds to an epitope in ECL-2 of CCR5, albeit in
a different region from that of 2D7 (202). This
antibody blocks chemokine binding, but is rel-
atively inefficient in blocking gp120 binding.
It would seem then that gp120 and the /?-che-
mokines interact with overlapping but dis-
tinct sites on CCR5. Chemotaxis and chemo-
kine signaling are not necessary for HIV-1
entry, and different receptor sites seem to
have distinct functions (203-205). As a gen-
eral rule, gp120 associates with both the N-
terminus and ECL-1 of the receptor (192,
195), whereas chemokines favor ECL-2 for
binding (206).
6.5.3 CCR5 Peptide Antagonists. Although
RANTES, MIP-la, and MIP-1/?have all dem-
onstrated an ability to suppress HIV infection
in vitro (541, there has been concern expressed
over using therapeutic agents that also func-
tion as agonists and have a potential to attract
leukocytes to various tissues. Chemokine de-
rivatives such as Met-RANTES, with an addi-
tional methionine at the N-terminus, and
AOP-RANTES, with an aminoxypentane that
is isosteric with the side chain of methionine,
are pure and partial antagonists of CCR5, re-
155
spectively. They inhibit HIV strains contain-
ing CCR5 at 10-fold greater potency than that
of the natural ligands (54). Combination ther-
apy has also proved effective at inhibiting
mixed infections (207). AOP-RANTES (CCR5
antagonist) and Met-SDFlb (CXCR4 antago-
nist), when used in combination, were much
more effective (9599%inhibition) at blocking
R5X4 dual-tropic HIV-1 primary isolates than
when used alone (32-61% inhibition).
Truncated peptides have also been evaluated.
A truncated version of RANTES (RANTES[s-681)
binds to CCR5, yet is incapable of eliciting a
chemotactic response (53). This would tend to
support the role of the amino terminus in re-
ceptor activation. RANTES[s-681does block
HIV-1 infection in cells (208).
6.5.4 CCR5 Small Molecule Antagonists.
As a consequence of screening, the National
Cancer Institute chemical repository for in-
hibitors of HIV-1 replication, the distamycin
analog NSC65106 (42) was identified as hav-
ing antiviral activity (209). Its mechanism of
action appears to be through involvement of
chemokine receptors such as CCR5 and
CXCR4. It has also been shown to inhibit
CCRl and CCR3 and is inactive against CCR2
and CXCR2 (210). The monomer (43) and re-
gioisomer (44) were inactive against this panel
of receptors.
As part of an effort to identify CCR2b an-
tagonists, the ammonium salt (45) and the

phosphonium salt (46) were found to have fair
activity against CCR5 (IC,, values of 0.39 and
0.62 a, respectively) (211). Lead optimiza-
tion through ring expansion resulted in the
quaternary ammonium anilide (25) (CCR5
IC,, = 0.0014 a; CCR2b IC,, = 0.027 a).
Chernokine and Cytokine Modulators
Also known as TAK-779, (25) is inactive
against other chemokine receptors such as
CCR1, CCR3, CCR4, and CXCR4 (202).
TAK-779 inhibits the binding of anti-CCR5
mAb 45531 to MOLT-4/CCR5 cells and is
therefore presumed to bind to ECL-2 (202).
However, it does not inhibit binding of 2D7,
which maps to a different region of ECL-2; or
of 3A9, which recognizes the amino terminus
of CCR5 (123). TAK-779 is an effective inhib-
itor of HIV-1 in T-cells and blocks R5HIV-1
replication without cytotoxicity to host cells
(212,213).
The binding site of TAK-779 has been ex-
plored using Ala-scanning mutagenesis on
more than 60 residues in the N-termini and
extracellular loops 1-3, and more than 35 res-
idues within TM1-TM7 (214). In this study
they could not identify specific amino acid res-
idues within the extracellular domains of
CCR5 that affected the antiviral action of
TAK-779. However, several residues in the
TM domains (L33, Y37, T82, W86, Y108,
T123, and E283) were found to interact with
TAK-779. The binding site of TAK-779 has
been postulated to be within a cavity formed
between helices 1, 2, 3, and 7 of the CCR5
The sulfonyl spiropiperidine (47) has also
demonstrated activity against CCR5 (IC,, =
35 nM)(215). The importance of an enantio-
selective interaction is suggested in that the
(2R)diastereomer of (47) has an IC,, value of
870 nil4 for CCR5, and is therefore 25 times
less potent than the (2s) isomer. HIV-1 viral
replication in PBMCs is inhibited by (47), with
a modest IC,, value of 6 a. The SAR of
the C-2 phenyl fragment has also been inves-
tigated, with the 3'-C1 derivative proving
optimal (CCR5 IC,, = 10 nM) (216). Oral bio-
availability within this series of sulfonyl spi-
ropiperidines has proved to be very poor (F,,
Constrained analogs have also been pre-
med. The 3,4-trans-pyrrolidine (48) shows
etter potency (CCR5 IC,, = 26 nM) than the
esponding cis analog (CCR5 IC,, = 3500
iarylsulfones (49) and (50) were identi-
ed as early leads in a Schering CCR5 program
218-220). These antagonists had been ini-
tially prepared for a muscarinic program and
showed significant activity against the M2 re-
ceptor (49): CCR5 Ki= 1.0 @, M2 Ki= 1.3
nM, (50):CCR5 IC,, = 440 nM, M, Ki= 0.8
nM).
The initial strategy to improve potency and
selectivity focused on substitution on and
around the piperazine (218). Chirality of the
piperazine 2-methyl was used to improve po-
tency and selectivity, as demonstrated by the
preference for the (2s)-methyl piperazine (51)
(CCR5 Ki= 28 nM, M, Ki= 485 nM). The
absence of any substitution at the 2-position
(52) resulted in a dramatic loss of activity
(CCR5 IC5, = 1300 nM).
Additional SAR andyses found that re-
placement of sulfonyl with methylene, as in
(53), retained potency and selectivity (CCR5
Ki= 18 nM, M, Ki= 760 nM). The next con-
cern was metabolic lability of the methylene
dioxy, and the 3'-chlorophenyl analog (54) was
found to be a suitable alternative (CCR5 Ki=
45 nM, M, Ki= 1400 nM). Both (53) and (54)
were shown to block viral entry at subcyto-
toxic levels (IC,, values of 1.7 and 12 nM, re-
spectively). In addition, (53)inhibited the rep-
lication of primary HIV-1 isolates in
peripheral blood mononuclear cells (PBMCs)
(IC,, = 8 nM).
158 Chemokine and Cytokine Modulators

Further exploitation of these leads led to M, Ki = 1323 nM) (220).However, this com-
the discovery that the shortened piperidino- pound has poor biovailability, presumably be-
piperidine amide analog (55) also shows sig- cause of metabolism at the benzylic site. TO
nificant inhibition of CCR5 (CCR5 Ki = 66 nM, address this shortcoming, ethoxime was intro-
6 CC Receptors
luced, followed by subsequent replacement of
he then labile 2,6-dimethyl phenyl with a
yridine N-oxide fragment, to give (56). This
d t e d in signliicant improvements of rat
h m a levels a€ter oral administration (CCR5
6 = 45 nM,M, Ki = 1400 nM, rat pk at 10
n&g p.0.: AUC,, h) = 8200 ng mL-l'h-',
P(mt, = 63%,F(monkey) = 52%). The piperazine
do^ (57) also showed im~roved~harrnacoki-
I--- -~~ 1-
~eticscS(~~R5K,= 7 nM,M, K~= 250 0, rat pk:
AUCo-, h) = 9243 ng mL-"h-l). Compound
(56) has also been referred to as SCH-C, and is
wrted to have entered Phase I clinical trials.
3pwif1cchemokine receptors involved in den-
&tic cell biology are a topic of current inves-
tigation. Origmally cloned as STRL-22, CKR-
L3, GPR-CY4, or DRY6, CCR6 has only one
chemokine ligand, MIP-3a (CCL20) (221-223).
More recently, it has been shown that a family
of antimicrobial peptides, the /3 defensins, also
bind CCR6, although their affinity for this re-
ceptor is significantly lower than that of
MIP-3a (224). CCRG has been shown to be ex-
pressed in PBMCs, and in a subpopulation of
immature dendritic cells. CD34+-derived den-
dritic cells, but not monocyte-derived den-
dritic cells, show CCRG expression and a sig-
nificant response to MIP-3a (225). Recently,
Yang and colleagues (226) have shown that in
the presence of TGFP, monocyte-derived den-
dritic cells are also able to express CCR6. In
addition, CCR6 has been found to be expressed
in a subset of memory T-cells as well as in a
subset of B-cells (227, 228). Moreover, it has
been described that CCR6 plays an essential
role in the arrest of a subset of memory T-cells
to activated dermal endothelium under physi-
ologic flow conditions (229). All these results
strongly support a role for MIP-3alCCR6 in
dendritic cell migration and in the recall im-
mune response.

CCRGP1 mice have been recently gener-
ated by two different groups. In the Cook
study, there was no evidence of gross abnor-
malities in any major organ, and all major leu-
kocyte populations appeared normal (230).
Dendritic cells coexpressing CDllb and
CDllc were absent from the subepithelial
dome of Peyer's patches. Some decreased hu-
moral response to orally administered rotavi-
rus was observed but no important delay in
the viral clearance. In a second study, using a
contact hypersensitivity model and a delay hy-
persensitivity model for the analysis of
CCR6-I- mice, Varona and colleagues de-
scribed a role for CCR6 in the activationtmi-
gration of CD4+ T-cell subsets (231). More
recently, it has been reported that CCR6-I-
mice are protected from developing allergic
lung inflammation and airway hyperreactivity
in a cockroach model (232).
CCR7 expression has been detected in mature
dendritic cells (225, 233, 234)' nahe T-cells,
and a subset of memory T-cells (central mem-
Chemokine and Cytokine Modulators
ory T-cells) (235, 236). Two ligands for CCR7
have been identified: MIP-3P (CCL19) and
SLC (CCL21) (237). CCR7 and CXCR5 to-
gether have been proposed as the main recep-
tors involved in cell recruitment into the
lymph nodes, Peyer's patches, and spleen. The
constitutive expression of MIP-3P (ELC) and
SLC in lymphoid tissue supports a role for
these chemokines in the trafficking of lympho-
cytes to secondary lymphoid organs. MIP-3P is
also expressed in interdigitating dendritic
cells, whereas the other ligand for CCR7, SLC,
is secreted by stromal cells (238-240). Dieu
and colleagues (225) have shown an interest-
ing relationship between CCRG and CCR7 ex-
pression in dendritic cells. Although CCR6 is
expressed in immature dendritic cells and
these cells are responsive to MIP-3a, cell mat-
uration causes CCRG downregulation and
CCR7 upregulation. Concomitant with the ex-
pression of CCR7, mature dendritic cells ac-
quire the capability to respond to MIP-3P.
Other studies have also observed CCR7 up-
regulation in mature dendritic cells (233,234).
The differential -expression of these chemo-
7 CXC Receptors
kine receptors likely accounts for the distinct
migration patterns observed for immature
and mature dendritic cells.
A natural mutation in mice, the plt muta-
tion, has helped to elucidate the role of CCR7
and its ligands. Mice homozygous for the mu-
tation show impaired homing of T-cells to the
lymph nodes and spleen (241). These mice also
show a decreased number of interdigitating
dendritic cells in lymph nodes. Recently, the
plt mutation has been associated with a dele-
tion that results in the lack of MIP-3P and
reduced expression of SLC (242). The pheno-
type of plt mice reflects the role of CCR7 in
dendritic and T-cell recruitment.
CCR7 knockout mice show a significant re-
duction in ndive T-cells in secondary lymphoid
tissues as well as important lymphoid tissue
abnormalities. When challenged, CCR7-'-
mice show an impairment in T-cell-mediated
immune response, including impairment of
delayed-type hypersensitivity reactions (243).
Because of the importance of CCR7 in T- and
dendritic-cell recruitment, targeting CCR7
might represent a viable way of interfering
th acquired immune responses. However,
ven that no studies have been reported in
ich the role of CCR7 has been specifically
tudied in animal models of human diseases, it
difficult to predict from the in vitro data and
few in vivo studies available, the conse-
uences of inhibiting CCR7.
CR8 was cloned by several groups as an or-
han chemokine receptor (TER1, ChemR1,
CKR-L1) (244-246). CCR8 is constitutively
expressed at high levels only in the thymus,
although low levels of mRNA are also present
in the spleen (244). A ligand for human CCR8
has been shown recently to be the CC chemo-
kine 1-309 (CCL1). It was the only chemokine,
among a large panel, to induce intracellular
calcium mobilization and chemotaxis in
CCR&transfectedcells (247,248).
Both CCR8 and CCR4 are preferentially ex-
ressed by Th2 cells (165-167, 249) and 1-309
ces a preferential migration of human
h2-polarized cells in vitro (167, 249).
A murine homolog of CCR8 has been char-
erized and shows a pattern of expression
slmilar to that of hCCR8 (250). In the periph-
161
ery, CCR8 mRNA expression was present only
in a Th2-polarized subset of CD4+ T-cells.
The ligand for mCCR8 has been identified as
T-cell activation gene (TCA)-3 (250, 251).
TCA-3 induces chemotaxis and activation of
neutrophils, macrophages, mesangial cells,
and vascular smooth muscle cells (252-255).
CCR8 knockout mice have been generated
and analyzed in a mouse model of allergic lung
inflammation (256). In models of Th2-type re-
sponse, including Schistosoma mansoni-solu-
ble egg antigen (SEA)-induced granuloma for-
mation as well as ovalbumin- and cockroach
antigen-induced allergic airway inflamma-
tion, CCR8 knockout mice showed impaired
Th2 cytokine production and reduced eosino-
phi1 recruitment. By contrast, in a typical T h l
model of secondary granuloma formation,
CCR8 knockout mice behaved in a manner no
different from that of wild-type mice. These
results suggest an important role for CCR8 in
Th2 functional responses in vivo.
7 CXC RECEPTORS
7.1 CXCRl and CXCR2
Two human receptors for IL-8 (CXCL8) have
been identified. CXCRl preferentially binds
IL-8 and GCP-2, whereas CXCR2 binds all
ELR-containing chemokines, including IL-8,
GROa, GROP, GROy, NAP-2, ENA 78, and
GCP-2 (18, 257-259). IL-8 is primarily re-
garded as a neutrophil chemoattractant, in
spite of the fact that these receptors are ex-
pressed on eosinophils, monocytes, and ba-
sophils (18). CXCR112 activation, in addition
to inducing chemotaxis in different leuko-
cytes, also stimulates the release of granule
enzymes and respiratory burst in neutrophils
(260).
A close association has been described for
IL-8 and other CXCRlI2 ligands in acute in-
flammation, including neutrophil-mediated
inflammatory diseases such as ischemia-
reperfusion injury, bacterial pneumonia,
adult respiratory distress syndrome, and
other infectious diseases. Neutralization of
IL-8 in rabbits has shown dramatic protection
in several models of neutrophil-mediated
acute inflammation (261,262).

The exact role of CXCRl and CXCR2 has
been difficult to define, in part because mice
have a single receptor for IL-$-like molecules.
Disruption of this receptor gene in mice re-
sults in abnormal myeloid and lymphoid de-
velopment (263). The mice fail to mobilize
neutrophils in response to intraperitoneal
thioglycolate challenge. In an allergic model of
lung inflammation, mice with a targeted dele-
tion of the IL-8 receptor showed a marked de-
crease in the recruitment of neutrophils into
the airways (264). After multiple challenges,
knockout mice had increased B-cells and fewer
neutrophils compared to those of wild-type
(wt) mice. A diminished hyperresponse to
methacholine was also observed in the knock-
out mice, suggesting that the IL-8 receptor
can modulate the IgE response and bronchial
hyperresponsiveness, in addition to the regu-
lation of neutrophil infiltration. In support of
its possible role in airway disease, several
studies have shown the presence of IL-8 in the
bronchoalveolar lavage (BAL) fluid of asth-
matic patients (265-268).
In addition to their role as chemoattrac-
tants for neutrophils, CXCRl and CXCR2 li-
gands have been shown to be potent angio-
genic factors, whereas most non-ELR-CXC
chemokines (CXCR3, CXCR4, and CXCR5 li-
gands) are angiostatic (269, 270). Several ex-
amples of diseases in which the balance be-
tween angiostatic and angiogenic chemokines
is altered have been described, including
chronic pancreatitis, inflammatory bowel dis-
ease, psoriasis, and idiopathic pulmonary fi-
brosis (271-273).
7.1.1 CXCRl/CXCR2 Receptor Structure.
CXCRl and CXCR2 have a high degree of
amino acid identity (77%),and yet they show
different binding selectivity for CXC chemo-
kines. CXCR2 has similar binding affinity for
IL-8, GROa and NAP-2, whereas CXCRl is
selective and has high affinity only for IL-8,
and a much weaker affinity for GCP-2. It has
been shown that the N-terminus and ECL-2
are important for both ligand binding and se-
lectivity among CXCRl and CXCR2 receptors.
Ala scanning of the CXCRl extracellular do-
mains shows that several charged residues
(D11, R199, R203, D265, E275, and R280) are
critical for IL-8 binding (274-277). Similarly,
Chemokine and Cytokine Modulators
additional charged residues in the N-terminus
and ECL-2 of the CXCR2 receptor are also
shown to be critical for binding of IL-8, NAP-2,
and GRO to CXCR2. Residues important for
IL-8 binding are D9, E12, K108, and K120,
whereas E7, D9, and El2 significantly contrib-
ute to GROa binding (278). In this study it is
clear that residues contributing to the high
affmity binding site of IL-8 and GROa on
CXCR2 have both overlapping and uncommon
regions. Notably, mutations of the N-termi-
nus residues (D9, El21 and certain ECL-1 res-
idues (K108, N110, K120) reduce cell activa-
tion and receptor signaling to all three ligands
(278).
7.1.2 CXCR1/CXCR2 Antibodies. Neutral-
izing monoclonal and polyclonal antibodies of
CXCR2 have been generated to profile the
function of each IL-8 receptor (CXCR1 and
CXCR2), and important distinctions have
been proposed. Changes in calcium flux and
signaling for granule enzyme release can be
initiated in a chemokine-specific manner, and
blocked with antibodies specific to each recep-
tor (anti-IL8R1 and anti-IL8R2) (279). Respi-
ratory burst, activation of phospholipase D
(279), and neutrophil chemotaxis (280) are
proposed to be stimulated predominantly
through CXCRl. Priming of neutrophil
NADPH oxidase is also mediated primarily
through CXCRl(281). Understanding the role
of each receptor in contributing to a physio-
logic response of neutrophils will be important
in the design of new therapeutics.
7.1.3 CXCR1/CXCR2 Peptide Antagonists.
As indicated above, the chemokine N-termi-
nus seems to contain a critical recognition site
important for receptor binding and activation.
The ELR sequence is essential to CXCRlDi-
gand interaction and its importance in IL-8
has been established through analog deletion
or amino acid substitution within this region
(13). Truncation of the first five amino acids in
the IL-8 Nt (IL-8[6-721), or substitution of the
first five amino acids with two alanines (IL-
8[AAR7-721) gave high affinity antagonists ca-
pable of blocking receptor binding, neutrophil
chemotaxis, and respiratory burst (282). An
N-terminal truncation of GROa demonstrates
CXCR2 antagonism; similar changes in PF4
 7 CXC Receptors

have also been shown to antagonize CXCR2

(283).These NH2-terminallymodified analogs
had no effect on IL-&stimulated elastase re-
lease or superoxide generation, which are re-
sponses presumed to be mediated by CXCRl.
A naturally occurring inhibitor of IL-8 (IL-
8INH) has been identified in the supernatant
of polymorphonuclear leukocytes. This 52-kDa
protein blocks 1251-IL-8binding to the recep-
tor by binding specifically to IL-8. IL-8INH
demonstrates in uiuo activity by inhibiting
neutrophil infiltration to mouse ear (284).
Capped hexapeptides and heptapeptides
have also been reported as inhibitors of IL-8.
Antileukinate (58)inhibits the binding of IL-8
to both receptors and blocks both neutrophil
chemotaxis and activation (285). GROa and
IL-8 have been shown to be necessary for
growth of lung, stomach, and colon adenocar-
cinomas, and antileukinate will inhibit the
binding of GROa to specific receptors on ade-
nocarcinoma cell lines and block proliferation
(286). Antileukinate has also been shown to
block Staphylococcal enterotoxin A (SEA)-in-
duced neutrophil infiltration into the lung
(287).
disulfoxide (61) was also extracted from a ma-
7.1.4 CXCR1lCXCR2 Small Molecule An- rine organism, the South African asidian Lis-
tagonists. There are early reports of natural soclinum (289). All three natural products
products identified as CXCRl and CXCR2 an- (59-61) show modest affinity for CXCR2 (Ta-
tagonists. Extracts from sponge (Dysidea ble 4.9). However, similar activity against
, frondosa) provided Frondosins A (59) and C PKC was also noted, and thus the activity ap-
(60) as novel sesquiterpenes (288). Lissoclin pears to be nonselective.
164
Table 4.9 Inhibition of CXCRl, CXCR2, and PKCa with Frondosins A and C,
and Lissoclin Disulfoxide
CXCRla
Compound Binding
"IC5, values derived from competitive binding with 1261-IL-8.
bIC50values derived from competitive binding with lZ5I-IL-8.
The diaryl urea (62) inhibits lZ5I-IL-8bind-
ing to CXCR2 on CHO cells (IC,, = 500 nM)
(290, 291). A simple incorporation of a bro-
mine on one of the aryl groups (63) provided a
marked enhancement in CXCR2 &nity (IC,,
= 22 nM),with greater than 150-fold selectiv-
ity over CXCRl. This compound also inhibits
GROW and IL-&mediated chemotaxis of hu-
Chemokine and Cytokine Modulators
Ic60 (fl
CXCR2b
Binding
man and rabbit neutrophils. When adminis-
tered by i.v. infusion in rabbits, (63) blocks
IL-&induced neutrophil margination.
The contribution of the urea acidic group
has been evaluated by measuring binding as a
function of pH (292). Ureas bind strongly in
their anionic form, demonstrating as much as
a 10-fold improvement over the non-ionized
parent. Because the binding is reversible, the
mechanism of antagonism does not appear to
proceed through the isocyanate.
In an effort to identify an orally active drug,
further optimization in the diaryl urea series
led to (64),which inhibits 1251-IL-8binding to
CHOlCXCR2 membranes (IC,, = 39 nM)and
is selective against CHO/CXCRl (IC,, = 7400
nM) (293). The di&ylurea (64) also inhibited
GROa-induced Ca2+flux (IC,, = 5 nM), which
is CXCR2 mediated, while showing weaker in-
hibition against IL-Smediated Ca2+ flux (IC,,
= 426 nM), which is governed by both CXCRl
and CXCR2. Compound (64) was evaluated for
efficacy in an LPS-induced airway neutro-
philia within rabbits. When given orally 1 h
before and 4 h after an aerosol LPS challenge,
(64), at 25 mg/kg, showed significant inhibi-
tion of neutrophilia.
More recently, nicotinamide N-oxides such
as (65) were discovered to be antagonists of
 values of 0.09 and 0.28 a, respectively).
These compounds varied in their ability to
block GROa chemotaxis (IC,, values of >20
F
and 1.0 a, respectively). The discrepancy be-
tween binding and chemotaxis may reflect the
different binding interaction of GROa and
IL-8 on CXCR2 (277,278).

CXCR3 is a receptor for three CXC chemo-

1251-1L8binding to CXCR2 (IC,, = 1.0 $4)
(294). This compound acts as a functional an-
tagonist, as measured by its ability to blockboth
Oa-and IL-8-driven neutrophil chemotaxis.
oanilide to be
an important recognition element. Further
kines, IP-10 (CXCLlO), MIG (CXCLS), and
I-TAC (CXCL11) (see Table 4.1). This receptor
is expressed preferentially on T-cells and it is
upregulated upon activation of these cells.
The restricted expression of this receptor sug-
gests that its ligands are involved in the regu-

p
lead optimization improved ligand binding an- lation of lymphocyte recruitment observed in
tagonism, as indicated by (66) and (67) (IC,,.. autoimmune inflammatory lesions. IP-10 was
identified as a gene product induced by inter-
feron-y and expressed in delay-type hypersen-
sitivity reactions of the skin (295, 296). More
recently, CXCR3 has been described as being
present on all perivascular T-cells and astro-
F cytes associated with active lesions in multiple
/ N+\ sclerosis. In addition, CXCR3- and CCR5-ex-
0 pressing T-cells are enriched in the cerebro-
yo spinal fluid of patients with active disease
@% (297,298).
CXCR3-deficient mice show an impressive
response in an allograft survival model. In the
absence of any other treatment, allograft sur-
vival extended 55-60 days, whereas the nor-
(66) mal allograft survival for wild-type mice was
around 7 days. Expression of the three ligands
for CXCR3 has been reported in this model;
however, they show a sequential upregula-
tion, suggesting they may play a role at differ-
ent times during the development of the in-
F flammatory response (299).
CCR5 and CXCR3 have been identified as
being preferentially expressed on T h l cells
(300). Accordingly, IP-10 is approximately 10
times more potent on Thl cells than on Th2
cells. Because rheumatoid arthritis is classi-
fied as a Thl-type disease, it has been sug-
gested that CXCR3 could play a role in the
development of the disease. CXCR3-positive
T-cells and CXCR3ligands have been reported
in rheumatoid arthritis in the synovial fluid
(67) and synovium from these patients (301,302).

7.2.1 CXCR3 Antibodies. A CXCR3-spe-
cific monoclonal antibody (1C6) has been re-
ported (301). It blocks the binding of radiola-
beled IP-10 to activated T-cells (IC,, = 160
ng/mL), and its epitope is mapped to the first
15 amino acids of the receptor. IP-lO-medi-
ated chemotaxis was completely blocked by
use of 10 pg/mL of 1C6. Neutralizing anti-
serum against MIG and IP-10 has been shown
to be efficacious in transplant models, as has a
blocking anti-CXCR3 mAb in preventing
acute rejection (299). Similarly, anti-IP-10
mAbs increase survival times in a cardiac allo-
graft rejection model (303).
7.2.2 CXCR3 Small Molecule Antago-
nists. To date, there are very few reports on
small molecule antagonists for this receptor.
The 4-0x0-2,4-dihydro quinazoline (68) is re-
ported to block binding (IC,, < 0.8 a) of
IP-10 (304). There have been no reports of in
vivo activity with this type of antagonists.
As mentioned earlier, CXCR4 and CCR5 have
together been identified as the main corecep-
tors for HIV, acting with CD4 to enable viral
entry. More specifically, CXCR4 has been de-
scribed as an essential coreceptor for T-tropic
HIV-1- and HIV-2-mediated fusion (305,306).
CXCR4 was first cloned by several groups as
an orphan receptor named LESTR, HUMSTR,
or fusin (307). Later, SDF-la (CXCL12) was
identified as the only ligand for this receptor
(308,309). These studies have also shown that
SDF-la inhibits the infection of CD4lCXCR4-
expressing cells by T-tropic strains of HIV.
Chemokine and Cytokine Modulators
CXCR4 is widely expressed in different
blood cell types including B-cells, T-cells, and
monocytes (see Table 4.1). In addition, expres-
sion of this receptor has been described in non-
immune cells such as endothelial and epithe-
lial cells (307). SDF-la was originally isolated
from bone marrow stroma and it has been
shown that it supports the proliferation on B-
cell progenitors (310). Subsequent studies
demonstrate that SDF-la is also a potent che-
moattractant for lymphocytes and monocytes.
Unlike other chemokines, SDF-la is constitu-
tively expressed and is not regulated by proin-
flammatory cytokines (307,311).
CXCR4 knockout mice generated by sev-
eral groups have shown multiple functions as-
sociated with this receptor. SDF-la and
CXCR4 knockout mice present very similar
phenotypes. Both showed defective lympho-
poieis and myelopoiesis in the bone marrow
(312, 3131, as well as severe heart defects, de-
fective formation of large blood vessels, and
derailed cerebellar neuron migration (312-315).
Moreover, most CXCR4-'- mice died in utero
at 18.5 days of embryonic development and
the remainder died within 1 h after birth. This
is the only chemokine receptor null mutation
described to date that results in embryonic le-
thality.
7.3.1 CXCR4 Receptor Structure. Rat and
human CXCR4 chimeras suggest that ECL-2
of CXCR4 is the major determinant of receptor
binding to feline immunodeficiency virus
(FIV) (316). Mutation of the DRY motif and
the C-terminal tail of CXCR4 did not affect the
ability of the molecule to support fusion, sug-
gesting that neither signaling by way of G-
protein nor receptor internalization was re-
quired for fusion mediated by FIV (316). The
contribution of the negatively charged resi-
dues in the amino terminal of the CXCR4 re-
ceptor has also been evaluated (317). Point
mutation studies show that viral entry de-
pends on YDE-rich clusters in both the amino
terminus and the second extracellular loop of
CXCR4. Different viral isolates vary in their
dependency on residues in each domain. The
determinants of CXCR4 coreceptor function
are therefore more diffuse and isolate depen-
dent than those of CCR5 (317). Residues in
CXCR4 required for both SDF-1 binding and
7 CXC Receptors
signaling were identified as ECL2 (D187),
TM2 (D97), and TM7 (E288). The first resi-
dues (2-9) of the receptor N-terminus also
seem to be required for SDF-1 binding and
signaling. Residues important for ligand bind-
ing but not signaling in the N-terminus are
E13, E14, and Y21. The coreceptor activity of
CXCR4 (gp120 binding) was impaired by mu-
tations of two Tyr residues in the N-terminus
(Y7,Y12) and an Asp residue in ECL-2, ECL-3,
or TM2 (D193, D262, D97). These acidic resi-
dues may engage in electrostatic interactions
with basic residues of the a 1 2 0 HIV-1 enve-
lope protein in the V3 loop region (318). Ala
scanning of CXCR4 indicates that negatively
charged residues in the N-terminus (E14, E15,
and E32) and D97 in ECL-1 were important
for CXCR4 function as a coreceptor. It has
been shown that removal of the N-linked gly-
cosylation modifications of the CXCR4 mole-
cule permits the receptor to act as a potential
coreceptor for otherwise CCR5-dependent
HIV-1 Envs. Similar residues in the ECL-2 of
CCR5 were shown to be important for CCR5
coreceptor activity for isolates across several
clads. Together, these findings support the hy-
pothesis that there are conserved elements
important for coreceptor activity between the
CXCR4 and CCR5 molecules. These results
highlight a homologous and critical region in
ECLS for both CXCR4 and CCR5 and their
respective coreceptor activities (319,320).
7.3.2 CXCR4 Antibodies. A CXCR4 mono-
clonal antibody (12G5) was developed as the
sult of efforts to evaluate interactions of cel-
molecules and viral envelope glycopro-
s. 12G5was shown to inhibit infection and
fusion for isolates of HIV-2. After CXCR4
identified as a CD4-associated accessory
or for T-cell line tropic HIV-1 isolates,
G5 was shown to react with this protein and
owed specific binding for CXCR4 when com-
to CCR1, CCR2b, CCR3, CCR4, and
(321).
7.3.3 CXCR4 Peptide Antagonists. There
have been several reports on peptide antago-
ts that selectively block receptor binding
dlor activities of CXCR4. The importance of
N-terminal region of SDF-la was defined
ough use of truncated ligands. Short N-
terminal peptides have significant activity.
SDF['-'' could function as a weak agonist,
whereas a P2G mutation of this same trunca-
tion resulted in an antagonist (322). The N-
terminal residues form an important receptor
binding site with Lys-1and Pro-2 also contrib-
uting to receptor activation. Residues 12-17
(RFFESH) are important for receptor bind-
ing, but apparently do not contribute to acti-
vation. It is presumed then that this sequence
is involved in initial docking of ligand to recep-
tor. NMR-conformational analysis of these
truncated peptide ligands shows that residues
5-8 and 11-17 present as a p-turn P-aRtype
(323).
A 3D structure of the full-length peptide
SDF-1 was determined through use of NMR
(47), and important differences were noted,
when compared to other chemokine ligands, in
terms of hydrophobic packing and surface
charge distribution. Using a strategy that has
worked for other chemokine ligands, the
CXCR4 natural ligand, SDF-la, was derivat-
ized with methionine at the N-terminus. Met-
SDF-1P caused prolonged downregulation of
the receptor (3241, and as a single agent, or in
combination with other antivirals such as
Zidovudine or Nelfinavir, has activity against
H N - 1 isolates (325).
T22 (69) is an 18 amino acid analog of a
horseshoe crab blood cell-derived peptide. It
inhibits entry of T-tropic HIV-1 strains into
target cells. T22 blocks binding of 12G5 d b
to CXCR4 and inhibits SDF-1-induced Ca2+
mobilization. The structure of T22 mimics the
rigid structural conformation found in CXC
chemokines. The antiparallel P-sheet presen-
tation in SDF-1 is maintained within T22 by
virtue of its two disulfide bonds (326).
T22 is a cationic peptide characterized by
five arginine and three lysine residues. In T22
and its derivatives there appears to be a close
correlation between the number of positive

charges and anti-HIV activity (327). Ala scan-
ning shows Arg2, Nal3, Tyr5, and Argl4 to be
critical residues for activity (328). An electro-
static interaction of peptide with membrane
may contribute to cytotoxicity and a poor se-
lectivity index (SI = ratio of 50%cytotoxic con-
centration to 50% effective antiviral concen-
tration). Reducing the total positive charge
with P-citrilline(Cit) was hoped then to result
in less toxicity without a significant decrease
in anti-HIV activity. Thus, truncation and Cit-
substitution of T22 identified a 14-mer with a
single disulfide bond, T140 (70),as a more po-
tent antagonist (EC5,=3.3 nM, SI = 29,000).
In an attempt to further reduce the total pos-
itive charge, an additional (Cit) substituent
was introduced, resulting in TC14003 (711,
with a decreased net positive charge compared
to that of T22 and a greater selectivity index
(EC5,=4.0 nM, SI = 69,000) (328).
An arginine-rich polypeptide (72) has also
been described as an inhibitor of HIV-1 entry
acting through CXCR4. Again, the high posi-
tive charge on ALX40C is characteristic of the
p-sheet region of SDF-1 and these residues are
presumed to be a recognition element for
CXCR4 (329). The Tat peptide has also been
shown to inhibit HIV replication. The peptoid,
CGP-64222 (73), mimics the basic domain of
Tat, and has been shown to interact with
CXCR4 (330).
The binding site on CXCR4 is believed to be
localized to the second extracellular loop
(ECL-21,which contains a strong negative sur-
face charge with many anionic residues (326,
Chemokine and Cytokine Modulators
331, 332). The strongly cationic nature of
these peptide antagonists probably results in a
strong electrostatic interaction with the
receptor.
Noting the importance of the arginine, con-
jugates of L-arginine with aminoglycosides
have provided micromolar inhibitors such as
(74). They interact directly with CXCR4, as
evidenced by their ability to inhibit the bind-
ing of the 12G5 mAb (333).
7.3.4 CXCR4 Small Molecule Antagonists.
In 1992 bicyclams such as (75) were identified
as viricidal agents with activity against HIV-1
and HIV-2 (334).The bicyclam AMD2763 (75)
was shown to inhibit HIV replication in vari-
ous human T-cells. Based on time-of-addition
experiments, these agents were assumed to in-
teract with virus assembly after virus adsorp-
tion and preceding reverse transcription.
Their precise mechanism of action was not de-
termined until several years later, when the
anti-HIV replication activity of bicyclams was
shown to occur through direct interaction
with CXCR4 (331,335,336). This class of com-
pounds had no effect against M-tropic HIV vi-
ruses (CCR5-dependent). AMD2763 was
shown to bind with CXCR4 through competi-
tion studies with 12G5 and selectivity was
demonstrated by its ability to block Ca2+mo-
bilization stimulated by SDF-la, but not by
RANTES, MIP-la, or MCP-3.
A more potent analog, AMD3100 (76), in-
hibits HIV-1 replication at nanomolar concen-
trations (337). It is not toxic to host cells at 500
p H , and therefore shows a high selectivity in-
dex. Compound 76 binds to CXCR4, as deter-
mined by inhibition of 12G5, and does not
block 2D7 (the CCR5 mAb) (331). AMD3100
inhibits T-cell-&opic HIV-1 infection of PHA-
stimulated blasts from PBMCs, with an IC,,
value of 2-7 ng/mL, depending on viral strain.
This is a 10- to 50-fold improvement over
SDF1. Further derivatization of the bicyclam
series resulted in AMD3329, the py[iso-141ane
N4 dimer (77). Incorporation of heteroaro-
matics into the cyclam moiety lowered the
overall pK, compared to that of a secondary
amine. Compound 77 was determined to be
even more potent than AMD3100 in inhibition
of binding, Ca2+ flux, and suppression of X4
HIV-1 replication. The EC,, values against
 169

A A
NH2 H2N /;\ NH2 H2N /;\ N H ~ H2N f+7 N H ~
A
(72)

HIV-1and HIV-2 replication were 0.8 and 1.6
, respectively (338).
n an effort to improve the potential for
lbiting HIV-1 replication, bicyclam-AZT
gates (78) have also been described
The binding region for AMD3100 has been
defined through single amino acid substitu-
tions in the extracellular loops (ECLs) and TM
regions of CXCR4. ECL-2 contains five acidic
residues that have been implicated as an im-
portant recognition site of the V3 domain in

OH
ArgHN uNHAri
gp120. As part of the mechanism for viral en-
try, an electrostatic interaction is presumed
between the positively charged V3 loop (over-
all charge of i-8) of viral gp120 and the nega-
tively charged ECL-2 (net charge of -9) of
CXCR4 (332). Resistance to binding was con-
ferred by single amino acid substitutions in
ECL2. The putative binding interaction of
AMD3100 with the aspartic acids of ECL-2 is
Chemokine and Cytokine Modulators
consistent with the cationic nature of the bi-
cyclams and its array of eight proximal nitro-
gens that will form a multipositive charge
(340). The bicyclam unit seems to be very effi-
cient in disrupting this interaction. Through
site-directed mutagenesis it has been shown
that AMD3100 acts on the CXCR4 receptor
through binding to Asp171 in TM-IV and
Asp262 in TM-VI with each of its cyclam moi-
eties. Single or double substitution of these
aspartates by neutral asparagines resulted in
markedly decreased potency of AMD3100 for
inhibition of SDF-1 binding to the receptor,
SDF-1 induced intracellular calcium signal-
ing, and high affmity binding for certain HIV
strains. It is suggested that part ofAMD3100's
activity is attributed to conformational con-
straint imposed on the receptor by the rigid
connecting phenylene-bismethylene linker
(332, 341, 342).
The phannacokinetic properties of AMD3100
have also been evaluated and it has been
shown, not surprisingly, to have very poor bio-
availability (343,344). An unsuccessful effort
to improve the oral bioavailability through
ion-pair formation by use of sodium taurode-
oxycholate and sodium taurocholate as coun-
terions has been described (345).
AMD3100 has entered phase I clinical trials
as an i.v. formulation, and appears to be well
tolerated (346). A 20 mgkg dose provides a
median peak serum concentration of 118 ng;/
mL, which exceeds its 50% effective antiviral
concentration seen in cell culture (335-337).
8 VIRAL CHEMOKINES AND CHEMOKlNE
RECEPTORS
Herpesviruses, poxviruses, and lentiviruses
all encode chemokine and chemokine recep-
8 Viral Chemokines and Chemokine Receptors
fv?ON~
rGj"
NH 1 ( NH
LA
r-like molecules (347). Some of these mole-
es are structural homologs of chemokines
d chemokine receptors, whereas others are
ructurally unrelated but bind to either che-
okine or receptor and alter the function of
ese molecules. The selective advantage of
ressing these chemokine or chemokine re-
ptors is not yet clear. However, given that
emokines play a crucial role in organizing
host immune response, it is not surprising
t viruses have developed different strate-
es to interfere with the process.
Chemokine homologs are mostly encoded
herpesvirus and include three CC-type che-
okines, vMIP-I, vMIP-11, and vMIP-111.
P-I is encoded by HHV-8 and binds and
s calcium signals in T-cells through
. This same receptor also shows high af-
ty for vMIP-11. However, vMIP-I seems to
as an agonist for the receptor and vMIP-I1
aves as an antagonist (348). vMIP-I1 is a
oad-spectrum chemokine receptor antago-
. vMIP-111acts as a potent CCR4 agonist
attracts mainly Th2 T-cells.
Molluscum contagiosum virus (MCV) is a
man poxvirus that encodes a chemokine ho-
olog named vMCC-I (gene product of
148R)(349,350). This protein is related to
chemokines but the mature protein lacks
e amino acids in the N-terminus that are
tical for receptor activation. Thus, this mol-
le binds to several receptors such as CCR1,
R2, CCR5, CCR8, CXCR1, and CXCR2 but
not able to induce signaling, acting instead
an antagonist for these receptors.
In addition to chemokines, viruses also ex-
ss chemokine receptors. All of them bind
ltiple chemokine ligands but differ in their
ificity as well as in the signal transduction
pathways they engage. One interesting exam-
ple is the HHS-%encoded chemokine receptor
ORF 74. The HHS-8 virus has been implicated
in the development of Kaposi's sarcoma (KS).
ORF 74 encodes a constitutively active chemo-
kine receptor that binds several host CC and
CXC chemokines. It has been shown that this
receptor has angionenic, proinflammatory,
and oncogenic activities. When expressed as a
transgene in hematopoietic cells, it induces le-
sions that are histologically similar to those of
KS (351). This suggests that ORF 74 may play
a role in the pathogenesis of KS in humans.
The chemokine binding protein vCCI (pro-
duced by pox viruses) is a broad-spectrum CC
chemokine scavenger and completely inhibits
their function (350,352). Its strong inhibitory
properties are attributed in part to the high
affinity of vCCI for CC chemokines, having a
subnanomolar dissociation constant for all CC
chemokines. The affinity of CC chemokines is
greater for vCCI than for the native CC che-
mokine receptors. In a mouse model of allergic
lung inflammation, vCCI has been shown to
improve pulmonary function and to reduce in-
flammation (353). vCCI reduced significantly
the number of total leukocytes and eosinophils
in bronchoalveolar lavage fluid and lung pa-
renchyma. At the same time, a clear reduction
in airway hyperreactivity was observed. These
results not only show that intrapulmonary in-
hibition of chemokines by vCCI is a highly ef-
fective inhibitor of airway inflammation and
hyperreactivity in a mouse model but also sug-
gest a potential use of this agent for the treat-
ment of asthma.
The above examples represent just a small
group of all viral chemokine, chemokine recep-
172 Chemokine and Cytokine Modulators

Table 4.10 Cytokine Families and Some Examples (1)"

Family Cytokine Potential Disease Target
IL-I family IL-la RA, OA, COPD
IL-1p RA,OA, COPD
IL-18
bFGF
aFGF
Hematopoietic factors IL-2 Transplant
IL-3
IL-4 Asthma, allergy
IL-5 Asthma, allergy
IL-6 RA
IL-7
IL-9 Asthma, allergy
IL-11
IL-12 Autoimmume diseases
IL-13 Asthma, allergy
EPO
LIF
GMCSF
CNTF
IFNa Infectious diseases
IFNp Infectious diseases
IFN y Infectious, autoimmune diseases
TNF family TNF-a Arthritis, Crohn's disease, psoriasis
LT-a
LT-P
CD40L
CD30L
Fas-L
"CNTF, ciliary neurotrophic factor; EPO, erythropoietin; Fas-L, fas ligand; aFGF, acidic fibroblasts growth factor; bFGF,
basic fibroblast growth facor; GMCSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin;
LIF, leukemia inhibitory factor; LT, lymphotoxin; TNF, tumor necrosis factor; RA,rheumatoid arthritis; OA, osteoarthritis;
COPD, chronic obstructive pulmonary disease. For a more complete listing of cytokines see Ref. 1.

tor, or chemokine binding proteins. (For more
information on this topic, review Refs. 347,
350, 352.)
Cytokinesare extracellular signaling proteins,
mostly secreted; however, they could also exist
as membrane-bound proteins. They are pro-
duced by many different cell types and can
have an effect on adjacent cells (paracrine
fashion), at a distance (endocrine), or can act
on the same cells that produce them (auto-
crine). Cytokines mediate their action through
high affinity surface receptors and can demon-
strate a wide variety of actions, including the
coordination of inflammatory and immune re-
sponses. They are also involved in many stages
of the development of autoimmune diseases
such as rheumatoid arthritis, inflammatory
bowel disease, inflammatory skin diseases,
and allergic responses. Table 4.10 shows some
examples of the different cytokines and the
families to which'they belong.
Small molecule agents that inhibit the pro-
duction of cytokines have been described. For
example, glucocorticoids inhibit the synthesis
of several of these molecules by interfering
with the transcription factors AP-1 and NFKB
(354, 355). Among the cytokines downregu-
lated by glucocorticoids are IL-2, IL-5, IL-12,
and TNF-a. Because of the broad effects of
glucocorticoids, they have been used in the
treatment of many inflammatory conditions
such as rheumatoid arthritis, asthma, and
transplantation. Other negative regulators of
 okine production are cyclosporin and FK506.
se molecules exert their actions by inhib-
anscription factor nuclear factor of
activated T-cells (NFAT). Cytokines suscepti-
ble to inhibition by cyclosporin include IL-2,
IL-4, and IL-5. The inhibition of IL-2 produc-
nosuppressive effect
reason these drugs are being used
in transplantation therapy. Nonpeptidic small
molecule inhibitors that act through direct
binding of cytokines andlor their receptors are
ot common, given the large protein/protein
that would need to be disrupted.
Cytokine production can also be influenced by
modulation of various kinases, such as reduc-
gh p38 inhibition. It is beyond
o discuss all indirect
ne regulation. In this review
e concentrate on cytokines involved in im-
mune responses for which some modulators
ave been described in either animal models
is a proinflammatory cytokine that has
iple roles. It is a central mediator in en-
plays an important role in
ses such as rheumatoid ar-
tis. IL-1 is mainly produced by monocytes/
are activated by T-cells at
site of inflammation. B-lymphocytes and
ral killer (NK) cells can also be sources of
cytokine. Two IL-1 receptor agonists have
en described, IL-la and IL-1p. IL-1p is pro-
ced as a 31-kDa precursor and is processed
its mature form by IL-1p converting en-
(ICE) (356). Both IL-1 agonists possess
ivities; however, IL-1p is
creted molecule, whereas IL-la is primar-
ere are a large number of
es that are regulated by IL-1p. Among
d chemokines, cytokine
weptors, other inflammatory mediators,
p w t h factors, clotting factors, remodeling
factors, oncogenes, and adhesion molecules
(for a comprehensive list, see Ref. 357).
IL1 binds with high affinity to IL-1 recep-
in the recruitment of a
ond molecule, the IL-1R accessory protein
-1RAcP). This heterodimer is responsible
e signal transduction
de (358). The interaction between the
subunits results in the recruitment of MyD88,
an adapter molecule, which is then followed by
activation of IL-1R-associated kinase (IRA.)
(359). A second IL-1R has been described, IL-
1RII. This receptor does not signal; instead it
is shed from cells and acts as a decoy receptor
(360-362). IL-1RI is found on many different
cell types, including endothelial cells, smooth
muscle cells, epithelial cells, hepatocytes, fi-
broblasts, keratinocytes, and T-lymphocytes.
A natural regulatory molecule has been de-
scribed for IL-1, called IL-1R antagonist (IL-
1Ra). Usually this molecule is produced in
large excess with respect to IL-1 (363) and
binds to IL-1R with near equal affinity com-
pared to that of either IL-la or IL-lp, but does
not signal through the receptor (364). It has
been suggested that this molecule plays an im-
portant role in the regulation of IL-1p activity
in uivo.
9.1.1 11-1 Knockout and Transgenic Mice.
IL-1R knockout mice show a similar pheno-
type to that observed in IL-lp knockout mice
(365, 366). Both mutations have revealed the
role that IL-1 plays in IL-6 production and fe-
ver. In addition, IL-1R knockout mice had a
reduced delayed-type hypersensitivity (DTH)
response and acute-phase reaction to turpen-
tine. These mice showed an impaired ability to
fight infections by Listeria monocytogenes
(365). In addition, IL-1R is required for the
development of inflammatory lesions and clin-
ical symptoms in a mouse model of EAE (367).
9.1.2 11-1 Modulators/Clinical Data. Sev-
eral approaches have been used to antagonize
cytokine activity, including antibodies against
the cytokine or the cytokine receptor, mutant
forms of the cytokine that bind to the receptor
yet do not signal, and recombinant soluble re-
ceptors. The extracellular domain of the IL-
1RI has been used as a decoy receptor in sev-
eral animal models. For example, in a rat
antigen-induced arthritis model, administra-
tion of soluble IL-1RI reduced joint swelling
and tissue destruction (368). It also increases
survival of heterotropic heart allograft in mice
(369).
In the clinic, soluble IL-1RI pretreatment
before endotoxin administration shows no ef-
fect on fever or any other systemic effect. The

lack of efficacy of the IL-1RI treatment is
likely attributable to the fact that there was
already a decrease in IL-1P and IL-1% in
these subjects (370). A similar lack of efficacy
was observed when soluble IL-1RI was given
to rheumatoid arthritis patients. In a double-
blind, placebo-controlled trial, no significant
improvement in clinical scores was observed,
suggesting the inability of this agent to modify
the clinical course of the disease (371). In con-
trast to IL-1R1, IL-1RII has significantly
higher affinity for IL-1P than for IL-1Ra. A
soluble form of this agent could be more effi-
cacious as an inhibitor of the IL-1 responses,
although this would need to be proved.
Administration of recombinant IL-1Ra in
several models of chronic inflammation has
shown that receptor blockage by this molecule
reduces severity of the disease (372, 373).
When used in arthritis models, IL-1% inhib-
ited joint swelling and suppressed the inci-
dence of collagen-induced arthritis in mice
(374, 375). Moreover, IL-1% knockout mice
present a more severe disease phenotype com-
pared to that of wt mice, and even develop
arthritis or other inflammatory conditions
spontaneously, depending on the mouse strain
(376, 377). This suggests that an imbalance
between IL-1 and IL-1Ra may play a role in
the predisposition to local inflammatory dis-
eases. Recombinant human IL-1Ra has been
clinically evaluated in rheumatoid arthritis
patients (378,379). In a 6-month placebo-con-
trolled trial, 43% of the patients showed a 20%
improvement compared to 27% in the placebo
group. The IL-1Ra-treated patients also had
significantly less joint damage. In a study in
which IL-1Ra was given together with metho-
trexate to patients showing active signs of the
disease, a similar result was obtained. IL-1%
did not show significant side effects after 24
weeks of treatment; reactions at the site of
injection were the most frequently observed
adverse event. In summary, clinical studies
show that inhibition of IL-1P results in a sig-
nificant reduction in signs and symptoms of
rheumatoid arthritis as well as reduced joint
destruction. Recombinant human IL-1% has
recently been approved by the U.S. FDA for
the treatment of arthritis [Anakinra (Kine-
ret), Amgenl.
Chernokine and Cytokine Modulators
IL-2, also known as T-cell growth factor, is
produced by antigen-activated T-lymphocytes
and has a primary role in regulating T-lym-
phocyte proliferation and differentiation. Be-
cause T-lymphocytes can initiate and regulate
the immune response, IL-2 is assumed to have
a significant role in antigen-specific acquired
cellular immune response. IL-2 also activates
NK cells (380) and B-cells (381,382).Although
the effect on B-cells is somewhat controver-
sial, it does seem to have a clear role in tran-
scriptional activation of the J-chain and con-
sequent production of IgM (383). Because of
its effect on NK cells, IL-2 also has a role in
augmenting innate host defense. This cyto-
kine then may have therapeutic utility in
boosting immune reactivity in otherwise com-
promised patients, by promoting generation of
both innate and acquired immunity. The role
of the IL-2 receptor in human immune func-
tion is supported by the finding that patients
with X-linked severe combined immunodefi-
ciency (X-SCID)have genetic defects that map
to an IL-2 receptor gene locus (384).
IL-2 is a 15.5-kDa protein of 133 amino ac-
ids (385). A disulfide bond between Cys58 and
Cysl05 is essential for bioactivity, whereas
glycosylation does not seem to be important
(386). A 3D structure for IL-2 has been solved
to 3.0-A resolution (387), indicating that this
cytokine is composed of a fourfold bundle of
amphipathic antiparallel a-helices. A modifi-
cation of this structure has been proposed
based on secondary structure prediction and
comparison with other cytokines (388).
The IL-2 receptor (IL-2R) contains three
distinct membrane-associated subunits
(p55a, p75P, and p64y). Each subunit alone
will bind IL-2 with low affinity, but het-
erodimerization and heterotrimerization
bind IL-2 with increasing affinity. In addi-
tion to being on T-cells, IL-2R is also re-
ported to be expressed by B-cells, although
at levels 10 times lower than that of acti-
vated T-cells (389).
Signal transduction is linked to the cyto-
plasmic domains of the fly subunit (390, 391).
Activation of JAK and Src family kinases are
initial events for IL-2 signaling. These kinases
will phosphorylate ILSR and activate the
STAT and Ras-mitogen-activatedkinase path-
9.2.1 11-2 Knockout and Transgenic Mice.
The role of IL-2 in immune response has been
clarified in animal models by use of IL-2
knockout mice. Mutated mice that are defi-
cient in IL-2 production, generated by homol-
ation, display normal lyrnpho-
e development, but during the postnatal
mmunocompromised and die
thin the first few weeks (394,395). In a sep-
ate study, IL-&deficient mice with mutated
-2 genes were used to exam-
e the role of IL-2 in promoting clonal expan-
on of cytolytic T-lymphocytes during infec-
induced expansion of CD8+
-cells was inhibited, as was the production of
Ny by activated T-cells, leading to a reduced
ility to clear viral infection. It is worthwhile
oint out that mice deficient in CTLA-4,
is IL-2 dependent, display a
ar phenotype (397). IL-2 transgenic mice
e also been established and do not appear
(398). Notably,
mune disease in
9.2.2 11-2 Modulators/Clinical Data. An
al antibody has also been de-
ped. It prevents binding of IL-2 to its high
ty receptor and has been used therapeu-
nosuppressant in allograft
ection (399, 400).
Other negative regulators include small
uppressive agents that ex-
their effect through inhibiting production
activity of IL-2. For example, glucocorti-
production by inhibiting
ion factor c-jun (354) and
KB (355). Leflunamide and mycophenolic
thesis by inhibiting nu-
synthesis in T-cells. Cyclosporin,
, and rapamycin can suppress IL-2 gene
ion through inhibition of calcium-asso-
d signaling events regulated by cal-
longing to the NF-AT
ence is immunosup-
e of IL-2 expression
of these small mol-
ecule modulators demonstrate therapeutic
benefit and protection in patients undergoing
organ transplantation.
IL-2 has been evaluated therapeutically as
an enhancer of the immune system in the
treatment of both cancer and infectious dis-
eases. When a high dose of IL-2 is adminis-
tered over a 3- to 5-day period, severe systemic
toxicity, termed cytokine syndrome, is ob-
served. However, this therapy does provide an
antitumor response by an unestablished
mechanism in 15% of treated patients (401).
IL-2 may have therapeutic benefit in re-
storing immune function in AIDS patients. In-
dividuals infected with HIV are characterized
by a deficiency in circulating CD4+ T-cells
and, consequently, production of IL-2 is com-
promised. Macrophages infected with HIV-1,
and cultured in the presence or absence of
IL-2, show a diminished reverse transcriptase
activity in those cells treated with IL-2 (402).
In addition, the expression of CD4 and CCR5
(HIV-1 receptor and coreceptor, respectively)
was downregulated after treatment with IL-2.
Clinically, IL-2 has been used in the treatment
of HIV-1-infected individuals through either a
high dose treatment, as noted above for can-
cer, or a low dose continuous treatment regi-
men (403). Using this latter treatment, the
concentration of circulating CD4+ T-cells is
increased and the risk of opportunistic infec-
tion is reduced.
Th2-type cytokines such as IL-4, IL-5, and
IL-13 orchestrate a cascade of events during
development of an allergic inflammatory re-
sponse. This is demonstrated both clinically
and in preclinical animal models (404, 405).
IL-4 plays a critical role in the early commit-
ment of Tho cells to Th2 cells and regulates
IgE secretion by B-cells. It also induces
V-CAM expression on endothelial cells, pro-
motes eosinophilic inflammation, and in-
creases airway mucus production.
In asthmatic patients there is an increase
in serum and bronchoalveolar lavage fluid
IL-4 levels, and atopic individuals have a
higher frequency of IL-4-producing T-cells
(406, 407). Genetic studies have linked
asthma and atopy to the chromosome region
5q31-33, which includes IL-4, IL-5, IL-9, and

the IL-13 genes (408). Thus, aberrant produc-
tion of IL-4, or excessive response to this cyto-
kine resulting from genetic defects, might con-
tribute to the pathogenesis of asthma.
IL-4 is a 20-kDa secreted glycoprotein and
its expression is highly tissue specific. IL-4 is
produced by Th2 cells and natural killer cells
in response to stimulation through the T-cell
receptor (409). IL-4 binds to two types of re-
ceptor complexes, type I and type I1 receptors.
Type I receptor complexes are formed by the
IL-4 receptor a chain (IL-4Ra) and the com-
mon y chain (yC),which form part of the many
other cytokine receptor complexes. The type I1
receptor consists of the IL-4Ra chain and the
IL-13Ra chain. In both cases signaling occurs
through the JAWSTAT pathway, more specif-
ically through activation of STAT6 (410).
9.3.1 11-4 Knockout and Transgenic Mice.
-
In general terms IL-4 and IL-4Ra knockout
mice show similar phenotypes. Both develop
normally but they show a clear deficiency in
the Th2 response (411-413). They are able to
mount antibody responses but show a signifi-
cant decrease in their ability to generate IgE
and IgG1. In addition they show a decreased
capability to expulse intestinal parasites
(414).
Transgenic mice expressing IL-4 in differ-
ent tissues have been generated. When IL-4
expression is targeted to T-cells, they present
increased airway hyperreactivity, inflamma-
tion in the eye, and mild B-cell hyperplasia
(415). If IL-4 is targeted to the airway epithe-
lium, an enhancement of goblet cell hyperpla-
sia is observed (416). These experiments
showed the multiple functions 1 ~ - d c a exert
n
over different cell types.
9.3.2 11-4 Modulators/Clinical Data. Dif-
ferent approaches have been taken to neutral-
ize IL-4 activity, including soluble IL-4 recep-
tor, antibodies against IL-4, and mutated IL-4,
which acts as an antagonist of the receptor. In
a mouse model of airway inflammation, solu-
ble IL-4 receptor administered intranasally,
before allergen challenge, results in a reduc-
tion of eosinophil infiltration, V-CAM expres-
sion, and mucus hypersecretion (417). This
treatment, however, did not change airway
hyperreactivity in response to methacholine.
Chemokine and Cytokine Modulators
In another mouse model of allergic lung in-
flammation, antibodies against IL-4 adminis-
tered during sensitization reduce or abolish
airway eosinophilia and airway hyperreactiv-
ity (418,419). In similar mouse models, a mu-
tant form of murine IL-4 (Q116DN119D),act-
ing as both IL-4 and IL-13 antagonist, was
found to abrogate the humoral immune re-
sponse to allergen challenge, and completely
inhibited synthesis of allergen-specific IgE
and IgGl (420). Another murine IL-4 mutant
(deletion mutant C118), showing similar an-
tagonism against IL-4 and IL-13, inhibited the
development of airway eosinophilia and air-
way hyperreactivity (421). These results sug-
gest that a dual IL-4/IL-13antagonist could be
highly efficacious for the treatment of asthma.
Similar mutations have been described for hu-
man IL-4 and shown to be efficacious at inhib-
iting IL-4 and IL-13 responses in vitro (422).
As mentioned earlier, soluble receptors are
another means to antagonize cytokine activ-
ity. The soluble IL-4R (Nuvance, Immunex
Corporation) consists of the N-terminal region
of the IL-4Ra chain and has been shown to
bind IL-4 and sequester this circulating cyto-
kine. Nuvance was tested in a clinical trial
with promising results (423). The drug was
well tolerated and the placebo group showed a
decline in FEVl not observed in the treated
group. The efficacy of Nuvance TM was con-
firmed by improved asthma symptom scores in
the treated group compared to those of pla-
cebo. In spite of these promising results, Im-
munex had announced that Nuvance failed to
show efficacy in two Phase I1 clinical trials. A
third trial is still ongoing.
IL-5 is a hematopoietic growth factor cytokine
that plays a critical role in differentiation, pro-
liferation, activation, survival, and localiza-
tion of eosinophils (424). It is not detected at
high levels in healthy individuals. IL-5-in-
duced eosinophilia is characteristic of allergy
(424), parasitic infections (425, 426), and tu-
mors. Because eosinophils are a characteristic
feature of asthma and atopic dermatitis (4271,
and their numbers are reported to correlate
with severity of the disease, IL-5 is believed to
have a significant role in the pathogenesis of
atopic disease (424,428,429).
 In addition to having cell numbers associ-
ated with this disease, the biology of the eosin-
ophil also implicates this cell as a major con-
tributor to the disease process. Eosinophils
are phagocytic granulocytes that mature in
bone marrow, migrate to the blood stream,
and eventually localize at the site of injury.
They contain highly toxic components that are
released upon degranulation, leading to de-
struction of bronchial epithelium, mucosal
edema, and bronchial hyperresponsiveness
IL-5 is detectable in bronchial biopsies and
bronchoalveolar lavage (BAL) fluid of asth-
matics (433, 434). Additionally, inhalation of
IL-5 by asthmatics has been shown to cause
airway hyperresponsiveness (AHR)and an in-
crease in sputum eosinophils (435).
IL-5 is produced primarily by activated
CD4+ T-cells (436,437),and in lower levels by
eosinophils (438), mast cells (439, 4401, ba-
sophils, B-cells, NK cells (441,442), and endo-
thelial cells (443). The expression of IL-5 is
predominantly regulated at the transcrip-
tional level (444), and can be induced by a va-
riety of stimulants, usually through activation
of the T-cell receptor and a second signaling
pathway. IL-la and PMA can induce IL-5 ex-
pression; histamine can also increase the pro-
duction of IL-5 in activated T-cells (445).
Structurally, IL-5 is a disulfide-linked ho-
modimeric glycoprotein between 45 and 60
kDa (446). Glycosylation does not seem to be a
requisite for signaling; however, IL-5 must be
in native dimeric form for bioactivity (447).
Each monomer of IL-5 consists of four a-heli-
ces with an antiparallel p-sheet between op-
posing monomers. The monomers are main-
tained in dimeric form by cysteine bonds at
and 86 (448). Mutagenesis shows
s38, Lys39, and His41 in the sec-
lu89 and Arg91 in the P-strand;
d ThrlO9, GlullO, TRP111, and Is0112 in
fourth helix as being important contribu-
-5 interaction with the hIL-5Ra-
ain (449,450). Glu13 on IL-5 has been iden-
as a contact point for the p-chain of the
The IL-5R a-chain specifically binds IL-5
ty (451).When associated with a
cing p-unit that is also used by
r hematopoietin receptors such as GM-
CSF and IL-3, IL-5 binds with high affinity
(452-454). Both subunits are necessary for
signal transduction (455). The pathway for
signal transduction includes activation of two
Janus kinases (JAK1 and JAK2) and the sig-
nal transductionlactivator STAT5 (456,457).
9.4.1 IL-5 Knockout and Transgenic Mice.
Animal models have helped define the signifi-
cance of IL-5 and the eosinophil in the disease
process. IL5 administered to mice results in an
increase of eosinophils (458). Experiments
with IL-5-deficient and IL-5-transgenic mice
confirm a role for this cytokine in controlling
eosinophilia (459,460). In IL-5 knockout mice,
no eosinophils are produced in response to
parasite infection or sensitization with
ovalbumin, and there is minimal development
of lung inflammation or tissue damage. When
IL-5 expression is reconstituted in these mice,
pulmonary eosinophilia, tissue destruction,
and airflow limitation can be observed after
allergen challenge (459).
Transgenic mice that have constitutive ex-
pression of IL-5 with detectable levels of IL-5
in the serum and persistent eosinophilia have
also been described (461). These mice are de-
scribed as normal, which may suggest that ac-
tivation and degranulation of eosinophils may .
be necessary for disease pathology.
9.4.2 11-5 Modulators/Clinical Data. Modu-
lation of IL-5 can occur by inhibiting its pro-
duction and synthesis, or through direct bind-
ing to IL-5 receptor or ligand. Cytokines can
regulate IL-5 levels by inhibiting production.
For example, IFNy and IL-10 have demon-
strated they can inhibit IL-5 production in
uitro (462), whereas IL-12 indirectly modu-
lates IL-5 by biasing toward a T h l subset pop-
ulation (463).
Small molecule antagonists such as CsA,
FK506, and rapamycin all inhibit IL-5 produc-
tion (464,465). Glucocorticoids, in addition to
decreasing bronchial hyperresponsiveness,
can also downregulate IL-5 production (466-
468). OM-01 suppresses IL-5 protein produc-
tion, mRNA expression, and transcriptional
activity in PBMCs with no effect on either IL-2
or IL-4 (469, 470). (The structure for OM-01
has not been disclosed.)

A fusion protein of human IL-5R a-chain
and human IgG Cy3 chain (hIL5Ra-hy3) was
used for screening a library of low molecular
weight compounds, and the isothiazolone (79)
was identified as an IL-5R antagonist. By use
of radiolabeled isothiazolone, it was deter-
mined that association of (79) with the recep-
tor occurs through covalent modification of a
sulfhydryl group on a free cysteine (Cys66) in
IL5Ra (471,472).
In a separate study aimed at finding small
molecule antagonists, the soluble form of the
ligand binding a-chain of hIL-5 receptor was
used to identify (80) and (81)as inhibitors of
soluble IL-5R (IC,, values of 8 and 11 a,
respectively) (473,474).
These compounds also inhibited interac-
tion of IL-5 with the membrane-bound recep-
Chemokine and Cytokine Modulators
tor. They were found to be irreversible inhib-
itors, again suggesting a covalent interaction.
Not surprisingly, they also inhibited interac-
tion of the structurally similar IL-3 and GM-
CSF receptors with their corresponding
ligands.
Monoclonal antibodies to IL-5 have been
evaluated both preclinically and clinically. In
both monkey and guinea pig models of allergic
asthma, an anti-IL-5 mAb was able to reduce
lung eosinophils, airway hyperresponsiveness
(AHR), and lung damage in response to anti-
gen (475, 476). In a mouse model of asthma,
similar reductions in pulmonary and blood eo-
sinophilia were noted with the anti-IL-5 anti-
body (TRFK-5) (477). In this study, chronic
dosing did not affect normal immune function.
Similar results on peripheral blood eosinophil
counts were seen when a single injection of
anti-IL-5 receptor antagonist was given to
IL-5 transgenic mice (458). In mouse models
of eosinophilia induced by Nippostrongylus
brasiliensis, or Schistosoma mansoni and
Strongyloides venezuelensis, an anti-IL-5 anti-
body successfully reduced eosinophil respon-
siveness (478-480).
The humanized anti-IL-5 mAb, Sch 55700,
when given to Ascaris-responsive primates
(0.3 mpk i.v.1 also reduces pulmonary eosino-
philia (4811, and this biological response is
sustained for 6 months. In clinical trials, an-
other anti-IL-5 mAb, SB 240563 at 10 mpk i.v.,
was well tolerated and reduced circulating eo-
sinophils for up to 16 weeks (482). However,
there was no effect on the allergen-induced
late asthmatic response (LAR) or airway hy-
perresponsiveness (AHR) to histamine (483).
Sch 55700 was also unremarkable in a clinical
trial evaluatingsevere asthma patients (484).
These clinical data then suggest no association
between the concentration of eosinophils in
peripheral blood and sputum with the late
asthmatic response or bronchial hyperrespon-
siveness (BHR) that follows allergen chal-
lenge. Taken together with the results of IL-12
in a clinical setting (see below), the role of the
eosinophil as a contributor to disease progres-
sion in asthma is called into question.
IL-6 is a pleiotropic cytokine implicated in a
variety of biological activities, including im-
 ute phase reaction, bone re-
atopoiesis. This cytokine is
S, B-cells, monocytes, mac-
lid cells, fibroblasts, mast
0th muscle cells, glial
ondrocytes, keratinocytes,
phoblasts, mesangial cells, islet p-cells, and
oid cells (485-487), and can be induced by
li including LPS, IL-1,
Fa, IL-2, and PDGF (488-490). Abnormal
els of IL-6 can lead to several disease pa-
ologies and malignancies, including rheu-
matoid arthritis (RA), osteoporosis, myelo-
as, and lymphomas.
IL-6 can exist as five different molecular
21 to 28 kDa, based on
n and phosphorylation
he C-terminus and the
terminus appear to be important for elicit-
a biological response. As with other cyto-
es, IL-6 presents itself as a bundle of four
Similar to the IL-2 and IL-5 receptors, the
IL-6 receptor (IL-6R) contains two subunits,
an 80-kDa a-chain (IL-6Ra) and a 130-kDa
b-chain (gp130). The @chain can be shared
with the IL-11 receptor (493) and is the signal-
transducing unit of the IL-6R complex. IL-6
assembles the receptor complex in a sequen-
tial fashion, forming first a low affinity inter-
&ion with IL-6Ra, then a high affinity com-
plex with gp130 (494). IL-6 then induces a
transient tyrosine phosphorylation, which ac-
tivates the jun B transcription gene (495).
9.5.1 11-6 Knockout and Transgenic Mice.
The pathogenic role of IL-6 in disease is sub-
stantiated through the development and eval-
uation of IL-6 transgenic and knockout mice.
IL6 knockout mice are viable and under nor-
ha1conditions do not display any obvious phe-
ver, in response to
ction, IL-6 knockout
flarnmatory acute re-
ponse, and impaired
ation (412, 496, 497).
gen-depletion model intended
to induce an osteoporotic phenotype, IL-6-de-
6cient mice are protected from bone loss, sug-
has a function in regulating
8). More recently, a role for
in liver regeneration has been established
by use of IL-6-deficient mice. When sections of
the liver were cut away, the mice would dete-
riorate and die. However, when the mice were
pretreated with IL-6, hepatocyte proliferation
returned to normal and liver damage was pre-
vented. Thus, IL-6 therapy may be of benefit
in patients undergoing liver transplant, or
in those suffering from cirrhosis or chronic
hepatitis, which are characterized by liver de-
generation. The possible role of IL-6 in auto-
immune disease is made apparent with trans-
genic mice that overexpress the cytokine and
show an increase in agalactosyl IgG (499).
This immunoglobulin is increased in a variety
of autoimmune diseases such as RA, Crohn's
disease, type I diabetes, and pulmonary TB
(500-502). Transgenic overexpression of IL-6
in the CNS can lead to significant neurodegen-
eration and subsequent motor uncoordina-
tion, ataxia, and tremor (503).
9.5.2 11-6 Modulators/Clinical Data. Mono-
clonal antibodies to IL-6 have been described;
neutralization of IL-6 with one of these anti-
bodies (MH166) can inhibit human myeloma
cell growth in SCID mice (504, 505). In a pa-
tient with multiple myeloma, administration
of a murine anti-IL-6 antibody to human IL-6
blocked myeloma cell proliferation in the bone
marrow (506). An IL-6R antibody (PM1) has
also been described and shows a similar re-
sponse to MH166 in repressing tumor growth
in a SCID mouse (507). Anti-IL-6R antibodies
are currently in clinical trials for multiple my-
eloma and RA. The anti-IL-6 receptor anti-
body, MRA, has shown positive effects in an
open-label RA study (508, 509). Decreases in
tender and swollen joints were reported and
an ACR20 response was noted within 6 weeks
of treatment.
Receptor-binding sites on IL-6 have been
mapped by mutagenesis studies and critical
contact points with IL-6Ra and gp130 have
been identified. IL-6 mutants have been pre-
pared based on this information, leading to the
generation of "superantagonists" (Sant 1-7)
(510).
Small molecules such as Tenidap (511) and
retinoic acid (512) have been shown to inhibit
production of IL-6. Tenidap, in contrast to
other NSAIDs, suppressed production of IL-6
in vivo and, in a clinical setting, substantially

decreased the acute phase response in con-
junction with lower plasma IL-6 levels (513).
IL-12, originally termed cytotoxic lymphocyte
maturation factor (514), is a growth factor for
CD4+ and CD8+ T-cells and NK cells (515),
and has been shown to modulate the cytolytic
and proliferative activity of these cells. In
vitro, IL-12 is able to direct T-cells to a T h l
phenotype, and induces production of IFNy
and TNFa (516). IL-12 thus has potential to
promote an immune response, enhance the
host defense response (517), and treat Th2-
driven imbalances such as allergy or asthma.
Evidence for its role in protective immunity is
supported by the finding that individuals who
lack the ability to express a component of the
IL-12 receptor (IL-12Rp1) are susceptible to
bacterial infection (518, 519).
In contrast, IL-12 may contribute to Thl-
associated inflammation and autoimmune dis-
eases such as insulin-dependent diabetes mel-
litus (520), septic shock (521), multiple
sclerosis (522), and rheumatoid arthritis
(523). For these diseases, inhibition or neu-
tralization of IL-12 may have therapeutic
value (524).
IL-12 is a 75-kDa heterodimer with two dis-
ulfide-linked subunits (p35 and p40), and is
produced by macrophages (525), dendritic
cells (526), and airway epithelial cells (527).
Expression of IL-12 receptor (IL-12R) is lim-
ited to activated T- and NK cells. Functional
IL-12R has /3l and /32 subunits with p40 li-
gand segment binding primarily to /3l,
whereas the p35 ligand unit associates with
the p2 chain of the receptor (528). The /32 unit
appears to be responsible for signal transduc-
tion (529).
9.6.1 11-12 Knockout and Transgenic Mice.
IL-12 ligand knockouts have been developed
with mutations generated independently in
both the p40 and p35 genes. The mice are via-
ble, displaying normal development and nor-
mal levels of T-cells, B-cells, and macrophages
in the thymus, spleen, and lymph nodes (530,
531). In response to LPS, however, these mice
show an impaired ability to produce IFNy and
are unable to mount a T h l response.
Chemokine and Cytokine Modulators
9.6.2 11-12 Modulators/Clinical Data. En-
dogenous inhibitors of IL-12 include IL-4, IL-
10, IL-13, TGFp, and PGE,. Small molecules
such as pentoxifylline and acetyl salicylic acid
(ASA, aspirin) have also been shown to inhibit
IL-12 production in PBMCs and monocytes
stimulated with LPS. This inhibition is inde-
pendent of the endogenous inhibitors such as
IL-10, TGFP, and IL-4. Both aspirin and pen-
toxifylline inhibit accumulation of p40 mRNA
and do this through inhibiting the interaction
of NFKBin the p40 promoter region, leading
to suppression of the p40 gene.
The naturally occurring homodimer IL-
12p40 subunit acts as an antagonist of bioac-
tive IL-12 p35/p40 heterodimer. When IL-
12p40 homodimer is given to diabetes-prone
nonobese diabetic (NOD) mice, diabetes is
suppressed (532). This result supports a role
for IL-12 in driving Thl-driven autoimmune
diabetes.
Preclinically, IL-12 administration demon-
strates therapeutic benefit in models of infec-
tious disease (533, 534), cancer (535), and al-
lergic airway hyperresponsiveness (536,537).
IL-12's role in host defense is evidenced by the
protection that endogenous IL-12 provides
against viral infection. It does this through en-
hancement of NK and cytolytic T-lymphocyte
(CTL) activity (533). Recombinant IL-12 also
has a curative effect in mice infected with
Leishmania major (534). Because of IL-12's
ability to enhance NK and CTL activity, as
well as its potential to induce IFNy, this cyto-
kine may have therapeutic benefit as an anti-
tumor agent. IL-12 has shown potent antitu-
mor effects against a variety of murine
malignancies (535),and it is believed that this
effect is mediated through CD8+ T-cells.
IL-12 has entered clinical trials for cancer
(538-541), chronic hepatitis C (5421, and
asthma (543). Preliminary analysis shows
some positive benefit in patients with cutane-
ous T-cell lymphoma (541). To avoid severe
toxicity, an 1L-12 pretreatment regimen ap-
pears to be necessary (544).
Administration of IL-12 to patients with
mild allergic asthma significantly decreased
peripheral blood eosinophils and sputum eo-
sinophils (543). However, in contrast to the
mouse model, there was no clinical improve-
ment in airway hyperresponsiveness to hista-
 ne, nor was there an effect on the late asth-
atic reaction. As with anti-IL-5, which shows
similar clinical nonresponse, this calls into
uestion the role of the eosinophil in this dis-
ease, as well as the concept of asthma being
verned by a Th2 phenotype.
together belong to the
nes and share similar bio-
vities on human B-cells and mono-
. IL-13 is a 17-kDaglycoprotein pri-
ced by Th2 cells and, to a lesser
ent, by macrophages, dendritic cells, NK
Us, mast cells, and basophils.
The involvement of IL-13 in allergic disease
genetic data linlung IL-13 poly-
rphism to asthma. An association between
13 promoter polymorphism and increased
kof allergic asthma has been described (546).
an association between a
the IL-13 gene and serum IgE
his cytokine binds to two membrane-
und proteins, IL-13Ra1 (low affinity) and
affinity) (548). IL-13Ral re-
IL-4Ra to form a high
s complex is expressed in
hematopoietic and nonhema-
ietic cells. IL-13Ra2 alone is a high affin-
receptor but there is no clear evidence that
s protein is capable of transducing a signal
on IL-13 binding. Because of this, it has
it may act as a natural
13 function. Many of the com-
n functions assigned to both IL-4 and IL-13
able to the fact that they
alilL-4Ra high affinity com-
, IL-13Ral is not expressed
s not act on T-cells directly.
ever, it could modulate T-cell responses
ugh its effects on macrophages. Binding of
er cytokine to the IL-13RallIL-4Ra recep-
he activation of the JAW
Both IL-13 and IL-4 have been shown to
dion and IgE synthesis. It
so been reported that IL-13 acts on mac-
ages and regulates cell surface protein
ession. More recently, IL-13 has been re-
ed to induce gene expression in nonhema-
oietic cells, including fibroblasts, endothe-
lid cells, smooth muscle cells, and epithelial
cells (549, 550). Thus, IL-13 seems to have a
broader spectrum of actions on airway cells
compared to that of IL-4. For example, IL-13
but not IL-4 reduces p-adrenergic responsive-
ness of human airway smooth muscle cells
(551).
9.7.1 11-13 Knockout and Transgenic Mice.
The role of IL-13 in vivo has been studied by
use of transient or constitutive expression of
the cytokine. When IL-13 was expressed in an
inducible fashion in the lung, a very striking
phenotype was observed (552); mucus meta-
plasia, macrophage, lymphocyte, and eosino-
phil-rich inflammation and subepithelial fi-
brosis are characteristics shown by these
mice. These same features can also be found in
asthmatic patients. These experiments sug-
gest a role for IL-13, not only in the allergic
response but also in the remodeling and de-
struction of the airways. The role of IL-13 in
the development of lung allergic inflammation
in mice has been recently confirmed by the use
of knockout mice. Peripheral T-cells from IL-
13-'- mice produce fewer Th2 cytokines be-
cause of impairment of Th2 cell development
(553). In addition, IL-13 knockout mice do not
develop AHR, in spite of the presence of eosi-
nophil-rich inflammation. AHR was restored
in these mice by administration of recombi-
nant IL-13. These results indicate that IL-13
is necessary and sufficient for the induction of
AHR in mouse.
9.7.2 11-13 Modulators/Clinical Data. An
IL-13Ra2-IgGFc fusion protein was used to
assess the role of IL-13 in a mouse model of
allergic lung inflammation (554). The sys-
temic administration of this reagent into mice,
before the induction of lung inflammation by
ovalbumin, resulted in complete reversal of
airway hyperreactivity, even if treatment was
given after full development of the phenotype
(in contrast with IL-4). Blockade of IL-13 re-
verses the increase of mucus-containing cells
in this model. A human soluble IL-13 receptor
is currently being developed by the Genetics
Institute.
In a chronic model of Aspergillus fumiga-
tus-induced allergic asthma, immunoneutral-
ization of IL-13 has marked effects (555). The

use of anti-IL-13 antibody results in a signifi-
cant attenuation of airway hyperreactivity in
this model. When IL-4 is neutralized, no sta-
tistically significant decrease was observed. In
addition, neutralization of IL-13 inhibits col-
lagen deposition, subepithelial fibrosis, and
goblet cell hyperplasia. None of these effects
was observed when neutralization of IL-4 was
performed. Again, these results point to a
broader spectrum of effects for IL-13 com-
pared to that of IL-4, and for this reason IL-13
may be a more favorable target for asthma.
Other dual modulators for IL-4 and IL-13
are described in the IL-4 section of this review.
9.8 TNFa
TNFa was originally identified because of its
antitumor activity. However, later studies
have shown that this cytokine plays a major
role in autoimmune diseases and is also in-
volved in multiple activities, including metas-
tasis, viral replication, septic shock, inflam-
mation, and fever.
Human TNFa is a protein that exists in
both soluble (157 amino acids) and transmem-
brane form (233 amino acids). Soluble TNFa
is released from the cell membrane through a
TNF-converting enzyme and exists as a ho-
motrimer in aqueous solution (556,557). It is
produced primarily by monocytes/macro-
phages in response to various inflammatory
stimuli, but can also be produced by other cell
types, including T-cells, NK cells, dendritic
cells, and endothelial cells.
TNF mediates its action through two dif-
ferent receptors, referred to as p60 (p55, Type
I, CD120a) and p80 (p75, type 11, CD120D)
(558). In general, most TNF proinflammatory
activities are mediated through the p60 recep-
tor, whereas the role of the p80 receptor is less
clear. The crystal structure of the p60 receptor
bound to TNF has been solved (559). This
structure predicts that a ligand trimer brings
three receptor chains together to form a com-
plex. The p60 receptor is expressed by all cell
types examined to date. In contrast, the p80
receptor appears to be restricted to cells from
the immune system and hematopoietic cells.
The cytoplasmic domain of the TNF receptors
has been shown to bind to distinct serinelthre-
onine kinases and to cause phosphorylation of
the receptor (560).Activation of the p60 recep-
Chemokine and Cytokine Modulators
tor has been shown to activate apoptosis, the
nuclear transcription factor NFKB,and c-jun
N-terminal kinase (JNK) (561). NFKBactiva-
tion mediates many of the inflammatory activ-
ities of TNF, including the expression of cyto-
kines, chemokines, and cell adhesion molecules.
9.8.1 TNFa Knockout and Transgenic Mice.
The role of TNFa in vivo is not well under-
stood. It is believed that TNF is required for
protection against bacterial, fungal, parasitic,
and perhaps even viral infections and other
stressful stimuli (562). TNF knockout mice
are shown to develop normally. These mice
have normal thymus, but their spleen archi-
tecture is abnormal and their ability to fight
infection is reduced (563). They are also pro-
tected from lethal doses of LPS. Knockout
studies of the TNF receptor (p60) have shown
that mice deficient in this gene are resistant to
endotoxic shock but show increased suscepti-
bility to Listeria monocytogenes (564, 565).
9.8.2 TNFa Modulators/Clinical Data. In
human rheumatoid arthritis (RA), TNF pro-
tein has been observed in the synovial fluid,
and TNF mRNA in synovial cells, including
monocytes and macrophages (566,567). In an-
imal models of RA, anti-TNF therapy is highly
effective. These models include collagen-in-
duced arthritis, adjuvant arthritis as well as
streptococcal cell wall arthritis (568, 569). To
date, two different approaches have been
taken to neutralize TNF activity clinically: re-
combinant soluble receptor and monoclonal
antibodies against TNF. Etanercept (Enbrel,
Immunex) is a dimeric fusion molecule com-
posed of two p80 TNF receptor moieties bound
to an Fc portion of human IgG1. This molecule
is effective in both adult and juvenile RA (570-
572) and shows efficacy in controlling signs
and symptoms of the disease. In a randomized,
double-blind, placebo-controlled trial, 59% of
patients responded to Etanercept. The second
strategy consists in the generation of mouse
monoclonal antibodies against TNF. To de-
crease immunogenicity of the mouse antibody,
the Fc region is replaced by a human Fc do-
main. Infliximab (Remicade, Centocor) is a
chimeric antibody created by use of this strat-
egy and when used in combination with meth-
9 Cytokines
otrexate (to avoid the development of antibod-
ies), 52-58% response is seen in RA patients
(573,574).
The clinical effects of Etanercept are com-
parable to those of Infliximab. One advantage
of Etanercept is that it is given subcutane-
ously twice a week, whereas Infliximab is
given by slow intravenous infusion every 4-8
weeks. Another advantage is that Etanercept
also recognizes lymphotoxin, and this may ac-
count for its efficacy in juvenile chronic arthri-
tis. Currently, D2E7, a fully human anti-TNF
antibody, is being developed (Knoll). It ap-
pears to be effective in RA without the need for
methotrexate cotreatment (575).
Interferon y (IFNy) is produced mainly by a
subset of activated T-lymphocytes and natural
killer cells and is the main activator of macro-
phages (576). An increase in production of
IFNy is usually associated with effective host
defense against intracellular pathogens and
with autoimmune diseases. IFNy is a well-con-
served protein among animal species and its
biological active form is a 34-kDa homodimer.
However, it is heterogeneous in size because of
both enzymatic trimming of the C-terminus
and variations in glycosylation (577, 578).
The IFNy receptor consists of two sub-
units: the IFNGl subunit (IFNy receptor
a-chain, 90 kDa), which binds with high affin-
ity to IFNy; and the IFNG2 subunit (IFNy
receptor p-chain, 62 kDa), which does not con-
tribute significantly to binding but is neces-
sary for signal transduction (579). Both sub-
units are expressed ubiquitously on many cell
types. Upon stimulation, association between
the two subunits is induced and the signaling
cascade is initiated. The IFNy receptor activa-
tion process is through a JAWSTAT signaling
pathway, and JAK1, JAK2, and STAT1 are
specifically involved in mediating many IFN y-
dependent effects on cells (580, 581). The ef-
fects of IFNy are mostly at the transcriptional
level. Many genes are induced by treatment
with this cytokine. The genes that are induced
depend on the cell type and the presence of
other cytokines. For example, in macrophages
IFNy induces the expression of major histo-
compatibility complex (MHC) class I1 mole-
cules, contributing in this manner to antibody
183
formation and development of cytotoxic
T-cells (582). In endothelial cells, IFNy aug-
ments the production of the adhesion mole-
cule I-CAM, contributing to leukocyte infiltra-
tion during inflammation (583).
9.9.1 lFNy Knockout and Transgenic Mice.
The in vivo role of IFNy has been elucidated in
mice using null mutations for IFNy or the
INFy receptors (IFNG1and IFNG2). All these
mice present a common characteristic, in that
their ability to resist infection is greatly im-
paired. In these mice a significant reduction in
MHC class I1 molecules on macrophages was
observed (584-587).
INFy or INFy receptor knockout mice have
shown that,. depending.
A - on the animal models,
this cytokine can act as either an immunosup-
pressant or an immunopotentiator. For exam-
ple, IFNy receptor knockout mice are pro-
tected from developing autoimmune diabetes
in nonobese diabetic (NOD) mice (588). They
are also protected from developing inflamma-
tory bowel disease in an IL-12-induced disease
(589). In contrast, these knockout mice are
more vulnerable in other disease models, such
as the experimental autoimmune encephalo-
myelitis (EAE) model (590). Furthermore,
varied results can be obtained, depending on
the agent used to block IFNy actions and the
time in which the agent is delivered (see Mod-
ulators section).
9.9.2 lNFy Modulators/Clinical Data. Anti-
bodies against IFNy have been widely used in
animal models of human diseases. As indi-
cated for the knockout mice, the results of
blocking IFNy activity with antibodies de-
pends on the model used. One interesting sit-
uation is the collagen-induced arthritis model.
When the antibody is given early in the devel-
opment of the disease, a reduction in the se-
verity is observed. When the antibody is given
late, the result is disease aggravation (591,
592). Interestingly, knockout mice are more
sensitive to the development of the disease
(592).
Other experiments that use antibodies in
animal models have shown results similar to
those obtained with knockout mice. In disease
models of immune diabetes and inflammatory
bowel disease, anti-IFNy antibodies alleviate

the disease symptoms (593, 594). In disease
models of MS, such as EAE, anti-IFNy anti-
bodies enhance the disease (595-597).
Many research groups have recently iden-
tified patients with inactivating mutations of
IFNGRl or IFNGR2 who have a severe sus-
ceptibility to weakly pathogenic mycobacterial
species. Complete deficiency in IFNGRl re-
sults in infections with low levels of virulent
mycobacteria in early childhood (598).
Attempts to relate polymorphisms in the
human IFNy gene with disease association
have also been reported. In both Finnish and
Japanese populations, distinct IFNy gene
polymorphisms are found in insulin-depen-
dent diabetes mellitus patients, suggesting a
global disease association (599, 600). Recom-
binant human IFNy has been used in various
trials for the treatment of infectious diseases
such as chronic granulomatous disease, vis-
ceral leishmaniasis, and multidrug-resistant
tuberculosis. In all cases some benefit has
been reported after treatment with IFNy
alone or in combination with other drugs (601,
602). To date no clinical data have been pub-
lished regarding the efficacy of IFNy antago-
nists. However, Protein Designs Labs has a
humanized monoclonal antibody in phase I/II
trials for autoimmune diseases.
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Contents
1 Introduction, 204
2 Eicosanoid Biosynthesis, 205
3 Biochemistry of Leukotrienes, 205
3.1 Biosynthesis of Leukotrienes, 205
4 Sources and Actions of the Leukotrienes, 208
4.1 Discovery, Structure Elucidation (48), and
Cellular Sources of the Leukotrienes (491,
208
4.2 Biological Properties (781,209
5 Agents Inhibiting Leukotriene Biosynthesis, 210
5.1 Inhibitors of 5-Lipoxygenase (5-LO),211
5.1.1 Structure and Mechanism of 5-LO, 211
5.1.2 Antioxidants, 212
5.1.3 Iron Ligands, 212
5.1.4 N-Hydroxyureas, 213
5.1.4.1 in Vivo Activities and Clinical
Studies with N-Hydroxyurea
Inhibitors, 213
5.1.4.2 Second-Generation N-
Hydroxyureas, 214
5.1.5 Substrate Inhibitors, 215
5.2 Inhibitors of 5-LipoGgenase-Activating
Protein (FLAP), 216
5.3 Inhibition of LTA, Hydrolase, 219
5.4 LTC, Synthase, 220
6 Agents Antagonizing Leukotrienes, 220
6.1 Peptide-Leukotriene Antagonists, 220
6.1.1 Introduction, 220
6.1.2 FPL-55712-Derived Compounds, 221
6.1.2.1 LY171883 (Tomelukast),221
6.1.3 Leukotriene Analog Antagonists, 222
6.1.3.1 SKF 104353 (Pobilukast) and
SKF 106203,222
6.1.3.2 LY170680 (Sulukast), 222
6.1.3.3 Bayer ~7195,222

6.1.3.4 MK-0476 (Singulair,
Montelukast Sodium, 34) and
Its Precursors, 223
6.1.4 Peptide-Leukotriene Antagonists of
Diversified Structure, 224
6.1.4.1 O N 0 1078 (Pranlukast), 224
6.1.4.2 ICI 198,615 and ICI 204,219
(Accolate, Zafirlukast), 225
6.2 CysLTl Antagonist Clinical Studies, 226
7 Leukotriene B, Antagonists, 227
7.1 Introduction, 227
7.2 LTB, Antagonists Related to HAP-Type
LTD, Antagonists, 227
7.3 LTB, Antagonists Derived from the
Structure of LTB,, 228
7.4 Clinical Trials with LTB, Antagonists, 228
8 Biochemistry and Structural Biology of
Cyclooxygenases, 229
8.1 Prostaglandin Discovery and the Connection
to NSAIDs, 229
8.2 Cyclooxygenase 1,230
8.3 Cyclooxygenase 2,231
8.4 Structural Features of COX-1
and COX-2 Enzymes, 231
8.5 Biosynthesis of Prostaglandins, 232
8.6 Actions of Cyclooxygenase-Derived
Eicosanoids, 233
1 INTRODUCTION
A number of oxygenated products derived
from arachidonic acid, frequently termed eico-
sanoids, have profound physiological and
pathological effects. The history of these re-
views indicates the expansion of the aware-
ness of the medical importance of different
classes of eicosanoids. Two editions from this
series (1,2)contained one chapter listingpros-
taglandins as the only eicosanoid target. The
last volume broadly discussed both thrombox-
anes and leukotrienes together, while down-
playing prostaglandins by lack of detailed dis-
cussion. The current volume contains three
chapters on eicosanoid targets. Other chap-
ters describe prostanoid receptors and throm-
boxanes individually. This chapter describes
recent efforts in the COX-2 field and updates
the leukotriene review of the 5th edition (2).
The previous chapter on thromboxanes
and leukotrienes provided a thorough review
of both fields. However, a significant amount
of work has since been published in the area of
COX-2 Inhibitors and Leukotriene Modulators
8.6.1 Homeostatic Effects of Prostaglandins,
233
8.6.2 Pathophysiological Effects of Prosta-
glandins, 234
9 Selective COX-2 Inhibitors, 234
9.1 Agents Inhibiting COX-2,234
9.2 Structure-Activity Relationships, 234
9.2.1 4-Sulfonylphenyl COX-2 Class, 234
9.2.2 N-Arylsulfonamides, 241
9.3 Selective NSAIDs and NSAIDs Modified for
Improved COX-2 Selectivity, 242
9.4 Irreversible Inhibitors, 244
9.5 Biochemical Assays, Selectivities,
and Potencies, 245
9.6 In Vivo Models and Corresponding Efficacy,
246
9.7 Conclusion, 246
10 Agents Inhibiting COX-2 and 5-Lipoxygenase,
246
11 Clinical Efficacy and Safety of Selective COX-2
Inhibitors, 248
11.1 Osteoarthritis, 248
11.2 COX-2 and Cancer, 249
12 Conclusion and Summary, 250
13 Abbreviations, 250
leukotriene biology and modulators, thus
necessitating our editing and adding to the
previous review. In addition, a major break-
through in prostaglandin research occurred
with the discovery of a second cyclooxygenase
(PGHS-2 or COX-2) by several investigators.
This seminal work provided a new paradigm
and new medicinal and clinical research in an
area that seemed to be fully mature. The ap-
proval of selective COX-2 inhibitors Celebrex
and Vioxx are landmarks in eicosanoid re-
search. However, they are perhaps also just
first steps with the possibility that second-
generation COX-2-selective agents, mixed
COX-215-LO, selective PGE, receptor subtype
antagonists, or perhaps selective inducible
PGE, synthase inhibitors may ultimately pro-
vide improved therapy in inflammation, pain,
and perhaps cancer.
In this review, we first describe the paths
and enzymes leading to prostaglandins, leuko-
trienes, and lipoxins. We then take each of our
topics, separately dealing with leukotriene
modulators followed by cyclooxygenase inhib-
3 Biochemistry of Leukotrienes
Membrane Phospholipases*
phospholipids ~ipases
AA
Figure 5.1. Eicosanoid pathway.
itors. Each section contains a brief discussion
on the sources and actions of the mediators.
The efforts to develop biosynthesis inhibitors
andlor antagonists of these classes of media-
tors are then summarized with emphasis on
compounds that have progressed to clinical
evaluation. Because of the breadth of the area
covered by this chapter, it is impossible for it
to be fully comprehensive. Thus, we fre-
quently reference leading reviews in each sub-
heading area.
The eicosanoid metabolic pathway as it is cur-
rently understood begins with the liberation
of long-chain fatty acids from cell membranes.
In mammalian tissues the primary acid
released is arachidonic acid (AA, eicosatetra-
enoic acid, 0.620:4) (3). Some important ex-
ceptions to this generality are well known but
are outside the scope of this review. A number
of enzymes that release arachidonic acid have
been identified and are predominantly phos-
pholipases, although other enzymes clearly
are also important under some conditions and
in certain cell types (4-9). Of considerable im-
portance is the fact that under most condi-
tions free AA is normally kept very low by the
action of various acyl transferases (10). The
primary metabolic enzymes using AA as a sub-
strate to form oxygenated products are cy-
clooxygenases 1 and 2; 5-, 12-, and 15-lipoxy-
genases; and certain P450 enzymes.
,
HETEs
+ DiHETEs
Lipoxygenases ~ i ~ ~
COX-2
Prostaglandins
thromboxane
prostacyclin
3 BIOCHEMISTRY OF LEUKOTRIENES
After the release of AA from phospholipid
stores, the oxidative metabolism of AA occurs
through a variety of pathways (see Fig. 5.1).
Cyclooxygenase (COX) leads to the prosta-
glandins (11)and thromboxanes, whereas the
leukotrienes are formed through the 5-lipoxy-
genase (5-LO) pathway. In human cells, there
is a family of lipoxygenases, including 5-, 12-,
and 15-lipoxygenases, that bear significant se-
quence similarity to each other and that lead
to several series of linear metabolites. The
peptide and nonpeptide leukotrienes, various
hydroperoxy- and hydroxy-eicosatetraenoic
acids (HPETE~and HETEH) are products of
the 5-lipoxygenase LIP LO) pathway. LIP id^^
these eicosanoids, additional products are
made nonenzymatically (12) and some by dual
adion of 5-LO followedby &LO (lipoxins) (13).
3.1 Biosynthesis of Leukotrienes
The committed biosynthetic path to the leuko-
trienes begins (see Fig. 5.2) with the action of
5-lipoxygenase (5-LO) [EC 1.13.11.341 (14) on
arachidonic acid. Purified human 5-LO is an
unstable, 78,000-Da protein that has been iso-
lated and cloned (15, 16). It contains a tightly
bound, nonheme iron that is essential for en-
zymatic activity (17). Like all known lipoxy-
genases, 5-LO catalyzes the insertion of mo-
lecular oxygen into a 1,4-cis,cis-pentadiene
unit. The effect of 5-LO on AA is to abstract
stereospecifically the pro-S hydrogen at posi-
tion C, and to insert molecular oxygen at
 COX-2 Inhibitors and Leukotriene Modulators

Other
lipoxygenases

Other
/
HETEs
and diHETES
- Cell membrane
phospholipid pools

J-A_A
1 Phospholipase(s)

Arachidonic acid
I FLAP
COOH
Aooxygenases

Prostaglandins
thromboxanes

COOH COOH

COOH

LTA Hydrolase LTA Synthase

OH
- OH OH
COOH COOH

LTB 4

pGlutarnyl transpeptidase
OH OH
COOH COOH
4

Dipeptidase

LTE 4 LTD

Figure 5.2. Leukotriene pathway.

position C, to produce as an intermediate,
5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosa-
tetraenoic acid (BHPETE).The optimal activ-
ity of 5-LO requires the presence of Ca2+ (18),
ATP (19), and an accessory protein, 5-lipoxy-
genase-activating protein (FLAP) (20). 5-Li-
poxygenase also catalyzes the conversion of
5-HPETE to leukotriene A, [LTA,, 5(S),6(S)-
oxido-7,Wran.s-ll,14-cis-eicosatetraenoic
acid] through the stereospecific removal of the
pro-R hydrogen from position C,,, radical mi-
gration and formation of the 5,6-epoxide (21,
22). Alternatively, 5-HPETE can be reduced
to yield 5-HETE [5(S)-hydroxy-6,8,11,14-
(E,Z,Z,Z)-eicosatetraenoic acid].
Conversion of 5-HPETE to LTA, was orig-
inally thought to be attributed to a distinct
enzyme, LTA, synthase, but sequence cloning
studies showed that this protein is identical to
5-LO (23).
3 Biochemistry of Leukotrienes
A significant amount of work, much of it
from Merck and more recently the Peters-
Golden laboratory, has indicated that the
cellular localization of 5-LO is cell type and
stimulus dependent (24-29). Prominently cy-
tosolic in resting neutrophils, a number of
stimuli cause a fraction of the enzyme to asso-
ciate with nuclear membranes. In macro-
phages the enzyme is in the nuclear cytosol
and associates with the nuclear membrane on
activation. Control of this movement is associ-
ated with FLAP, as implied by inhibitor stud-
ies, but no specific binding of 5-LO to FLAP
has ever been shown (30). Nuclear responses
to leukotriene products are very likely the
topic of exciting future research. FLAP has
been proposed to present substrate to 5-LO
(31).
Further biosynthetic processing of LTA,
can occur along two distinct pathways. In the
first path, it is stereospecifically converted to
leukotriene B, [LTB,, 5(S),12(R)-dihydroxy- Lam (40) to speculate: "LTC, synthase may
6,14-cis-8,lO-trans-eicosatetraenoic acid] (32). represent a member of a lipid-binding protein
This conversion occurs by the addition of wa-
ter in a 1,8 manner across the conjugated trie-
nyl epoxide of LTA,, under the influence of the
cytosolic enzyme LTA, hydrolase [EC 3.3.2.61.
Because LTA, is an unstable epoxide, nonen-
zymatic hydrolysis can be easily accomplished.
However, this affords a complex mixture of
5,6- and 5,12-dihydroxyeicosatetraenoicacids
that contain no LTB,. LTA, hydrolase is a sol-
uble, zinc metalloenzyme (33), a monomeric
protein of molecular weight 70,000, that was
first purified from human leukocytes by Sam-
uelsson et al. (34).It has since been cloned and
sequenced from several sources (35). The hu-
man enzyme is 610 amino acids with a unique
sequence compared to that of other known ep-
oxide hydrolases.
Catabolic processing (metabolic inactiva-
tion) of LTB, occurs predominantly by se-
quential omega oxidation, first to 20-hydroxy-
LTB, by a cytochrome P450-catalyzed process
(34)and then to 20-carboxy-LTB, through a
different, soluble enzyme (36).
Alternatively, a second path for metabolic
processing of LTA, to the set of cysteinyl leu-
kotrienes begins by conjugation with reduced
glutathione (GSH), to form the parent sulfi-
dopeptide leukotriene C, [LTC,, 5(S)-hy-
droxy-6(R)-glutathionyl-7,9-trans-11,14-cis-triene metabolite is LTE, (47).
eicosatetraenoic acid]. This reaction is
catalyzed by another integral membrane pro-
tein, LTC, synthase [EC 2.5.1.371, that has
been localized to the perinuclear membrane
(37, 38). The purified l&kDa enzyme is very
unstable and requires Mg2+ ions and phos-
phatidyl choline for activity. Initially, it was
assumed that LTC, synthase was related to
the ubiquitous family of glutathione S-trans-
ferases. However, sequence comparison
showed little homology between LTC, syn-
thase and either the cytosolic or microsomal
GSH S-transferases (39). A detailed analysis
of protein sequence data showed that there
was 31% overall amino acid sequence identity
between LTC, synthase and FLAP, and that
in limited regions these proteins h e nearly
identical. This observation as well as the fact
that the FLAP inhibitor MK-886 inhibits
LTC, synthase activity (IC,,- a) 3 caused
family rather than classical GSH S-trans-
ferases."
Further metabolic progression of LTC,,
first to LTD, [5(S)-hydroxy-6(R)-cysteinyl-
glycyl-7,9-trans- ll,14-cis-eicosatetraenoic
acid], and then to LTE, [5(S)-hydroxy-6(R)-
cysteinyl-7,9-trans-ll,14-cis-eicosatetraenoic
acid], is by the sequential action of either
y-glutamyl-transpeptidase (41) or y-glutamyl-
leukotrienase (42) and cysteinylglycyl-dipepti-
dase (41).
Deactivation of the cysteinyl (peptidyl) leu-
kotrienes occurs by several pathways. The ox-
idative burst of activated phagocytes can inac-
tivate the cysteinyl leukotrienes (e.g., LTC,)
by the formation of inactive sulfoxides (43). In
this context, it is interesting that the sulfones
of LTC,, LTD,, LTE,, and LTF, have been
synthesized and were determined to be about
equally active in vitro with the parent sulfides
(44). Although isolation of the sulfones from
rat peritoneal cells has been reported (45), nei-
ther their biological significance nor their dis-
tribution is clear. In rat, N-acetylation and
omega-oxidation of LTE, occur to form the
N-acetyl-20-carboxy-LTE, (46). In humans,
however, the major excreted cysteinyl leuko-
 COX-2 Inhibitors and Leukotriene Modulators

4 SOURCES A N D ACTIONS OF THE in conjunction with Corey (57-60). LTA, was

LEUKOTRIENES recognized as the precursor to SRS-A and
LTC, was proposed, as a cysteine-containing
4.1 Discovery, Structure Elucidation (48),
derivative of 5-hydroxy-7,9,11,14-eicosatet-
and Cellular Sources of the Leukotrienes (49)
raenoic acid (59), to be the next member of the
biological cascade. Through further synthetic
Almost 60 years ago Feldberg, Kellaway, and efforts glutathione (60, 611, as earler sug-
Trethewie (50, 51) discovered that the addi- gested by the Parker laboratory (551, was
tion of cobra venom to perfused guinea pig identified to be the cysteinyl component, and
lungs caused the release of a slow-reacting, the absolute stereochemistry was assigned
smooth muscle contracting substance (SRS) (58,601. LTD, (61-64) and LTE, (63,65) were
that was distinct from histamine. Early stud- identified as natural SRS components after
ies on this mediator were usually carried out they had been synthesized by Corey's group as
by use of crude biological extracts and, as part of their effort to assign the structure of
such, the samples were contaminated by the LTC,.
presence of other physiologically active agents The name leukotriene was suggested for
(especially large amounts of histamine) that these compounds by Sarnuelsson et al. (64,66)
interfered with characterization of the SRS. In because they were found in leukocytes and had
the late 1950s and early 1960s Brocklehurst a triene-containing structure. The letters A, B,
worked extensively on the extraction, purifi- C, D, and E in part define progress along the
cation, and characterization of SRS (52). biosynthetic pathway and the numerical suffix
When he noted that SRS could be produced in was appended to describe the total number of
vitro from the lung tissue of previously sensi- double bonds found therein. Although the
tized guinea pigs upon antigen challenge, he products from arachidonic acid that are the
suggested that it be renamed SRS-A, slow re- subject of this review all contain four double
acting substance of anaphylaxis (53), in recog- bonds, analogous compounds, containing
nition that it was generated during anaphylac- three or five double bonds, may be biosynthe-
tic responses. sized starting from, respectively, 5,8,11-eico-
The minuscule amounts of SRS-A available satrienoic acid or 5,8,11,14,17-eicosapenta-
from natural sources and its chemical and bi- enoic acid (65, 67).
ological instability severely hampered efforts The cellular locations of leukotriene bio-
to characterize this mediator. In the 1970s a synthesis are determined by the distribution
breakthrough occurred with the combined and location of the enzymes needed for their
findings from the Piper (54), Parker, and Aus- production. 5-Lipoxygenase is found in only a
ten laboratories that: ( 1 ) under some circum- limited number of specific cell types, predom-
stances cyclooxygenase inhibitors caused an inantly of myeloid extraction. Because of this
increased production of SRS-A, which implied restricted expression, production of LTA, is
a link with arachidonic acid metabolism (55); largely limited to those cells (e.g., basophils,
and (2) HPLC could be used to purify SRS-A eosinophils, neutrophils, macrophages, mast
and that the UV absorbance of the HPLC Sam- cells, and monocytes) (66).These cells produce
ples implied the presence of a triene-contain- LTA,, further process it to either LTB, or
ing chromophore. LTC,, or secrete it.
Samuelsson and Borgeat's subsequent Neutrophils (67-69), monocytes, and mac-
work on arachidonic acid metabolism that led rophages are the major producers of LTB,
to the discovery of the blipoxygenase pathway (70). Once synthesized (e.g., by polymorpho-
(56) and to the assignment of the structure of nuclear leukocytes), LTB, is actively exported
(5S,12R)-dihydroxy-6,8,10,14-eicosatetra- by a carrier-mediated process (71). However,
enoic acid (55, 57) (later named LTB,) were LTA, hydrolase is a relatively ubiquitous spe-
critical to the efforts on SRS-A. Samuelsson cies found in almost all human tissues (72).
and Borgeat (56,58) proposed an unstable in- Because of this broad distribution, conversion
termediate (LTA,) as the precursor to LTB,. of extracellularly synthesized LTA, to LTB,
The structure of LTA, was correctly assigned can occur in a wide variety of cells. This has
4 Sources and Actions of the Leukotrienes
been exemplified with erythrocytes, which
lack 5-LO, but have the ability to augment
LTB, production when mixed with neutro-
phils (73). Also, enzymatic conversion of LTA,
to LTB, in human plasma has been demon-
The sulfidopeptide leukotrienes (LTC,,
LTD,, and LTE,) are synthesized by basophils
(75), eosinophils (67, 681, macrophages, and
mast cells. These cells generate the peptido-
leukotrienes because they have LTC, syn-
thase. Some other cells that lack 5-LO (e.g.,
platelets) have LTC, synthase and can convert
imported LTA, to LTC,. Like LTB,, LTC, is
actively exported to the extracellular environ-
ment (76). In this case export is by a probeni-
cid-sensitive membrane carrier-mediated pro-
cess that is temperature dependent and
saturable (77). Conversion to LTD, and LTE,
occurs extracellularly.
4.2 Biological Properties (78)
The biological effects of both LTB, and the
peptidoleukotrienes are mediated through
specific receptors (79,80). The effects of LTB,
are mediated through the BLTl (IUPHAR re-
ceptor nomenclature) and BLT2 receptors and
the peptidoleukotrienesare mediated through
the CysLTl and CysLT2 receptors.
Despite the fact that potent specific antag-
onists are available for these receptors, some
of which are approved drugs, only recently has
the molecular identity of LT receptors been
discovered. There are two known receptors for
TB,. These receptors differ in a f h i t y for
3H]LTB4by about 150-fold (81).Yokomizo et
al. (82) discovered BLTl using a subtraction
strategy from HL60 cells. This receptor corre-
sponds to the high affinity receptor and ap-
pears to be important in leukocyte chemotac-
tic response to LTB,. This laboratory also
discovered the lower affinity receptor BLT2
83) as did Kamohara et al. (84). The two re-
ceptors differ in expression with the high af-
Enity receptor expressed selectively in periph-
eral leukocytes, whereas the second receptor
is expressed in many tissues. The receptors
are structurally similar (45%amino acid iden-
tity). Interestingly, the BLT2 open reading
frame overlaps the promoter region of BLTl
(85). The BLT2 receptor is activated by both
12(R)-HETE and LTB, (86). Another less ex-
amined, but potentially important interaction
of LTB,, is with the nuclear receptor PPAR a
(87).
LTB, induces a broad range of proinflam-
matory responses. It is a potent chemotactic
and chemokinetic agent for eosinophils and
neutrophils from several species (88, 89). In
addition to influencing the directional migra-
tion of cells, it upregulates the production of
cell adhesion molecules, such as the a,-inte-
grin adhesion protein CDllb/CD18, that are
needed for cellular movement (90). It causes
accumulation of PMNs in viuo and at higher
doses can induce their degranulation (91),
with the resultant release of a broad array of
lysosomal enzymes (e.g., glucuronidase and ly-
sozyme) and the production of oxygen radi-
cals. LTB, increases vascular permeability
(92), the exudation of mucus, and membrane
permeability to calcium (93). McMillan and
Foster have hypothesized that LTB,, in con-
junction with other chemotactic mediators,
will have a synergistic effect on amplifying the
inflammatory response (94).
LTB, is viewed as an important mediator of
acute and chronic human diseases. It has been
hypothesized that agents that either block its
production or antagonize its effects might
yield an anti-inflammatory agent with im-
proved side-effect profiles compared to those
of current agents. Increased production of
LTB, has been detected in many inflamma-
tory diseases, including adult respiratory dis-
tress syndrome (ARDS), arthritis, asthma,
contact dermatitis, cystic fibrosis, gout, in-
flammatory bowel disease, psoriasis, and
rheumatoid arthritis (95).
Ironically, it was only after three cysteinyl
leukotriene antagonists had been approved
for clinical use that the two currently known
cysteinyl leukotriene receptors were identified
molecularly. However, two receptors were
proposed earlier by Labat et al. (96). The re-
ceptors were proposed to be seven transmem-
brane G-coupled receptors by several groups
(97). Metters, using photoaffinity methods in
guinea pig lung preparations, had also identi-
fied a molecular size for the CysLTl receptor
(98). The groups from Merck Frosst and
SmithKline Beecham identified the CysLTl
receptor in 1999. Using similar strategies, the
210
receptor was identified from screening tran-
siently expressed orphan G-coupled receptors
using LTD, to stimulate calcium flux (99).The
identified receptor expressed in HEK293 cells
had the reported preference for LTD, versus
LTC, in calcium mobilization and rank order
potency of specific antagonists. Localization
studies indicated expression in human lung
and bronchus and human peripheral leuko-
cytes as well as several other tissues. The re-
ceptor has 337 amino acids and shows some
homology to LBTl (28%) and P2Y receptors
(31%). Another receptor, the CysLT2 recep-
tor, was also discovered recently by three dif-
ferent laboratories (100). This receptor was
discovered through its sequence similarity to
the CysLTl receptor (36% amino acid iden-
tity). It had the reported equivalent potency of
LTC, and LTD, and was antagonized by
BAYu9773. This receptor has more limited ex-
pression that overlaps with the CysLTl recep-
tor but appears more cardiovascular and endo-
crine in nature. The gene has been localized to
chromosome 13q14close to a marker that has
been associated with atopic asthma in two
studies (100). Currently approved drugs have
activity only against the CysLTl receptor. It is
unclear what further pharmacological effects
an agent antagonizing both receptors would
have.
The cysteinyl leukotrienes are potent
smooth muscle constrictors both in vitro and
in vivo in human and animal studies. In hu-
mans LTC, and LTD, are approximately 1000
times more potent than histamine, on a molar
basis, in inducing contractions on isolated hu-
man bronchus. LTE, is about 1/10 as potent as
LTC, or LTD, (100). Inhalation studies in hu-
mans have also shown that the leukotrienes
are about 1000 times more potent than either
histamine or methacholine in producing bron-
choconstriction (101). The sensitivity to in-
haled LTD, is such that the order of respon-
siveness is asthmatics > allergies > normals
(102). Inhaled leukotriene D, induces hyper-
responsiveness to methacholine (103) and in-
halation of LTE, induces eosinophil migration
into the lungs of asthmatic test subjects (104).
Because eosinophilia has been correlated with
the degree of asthma severity, it is possible
that the leukotrienes play a significant role in
COX-2 Inhibitors and Leukotriene Modulators
converting the initial acute bronchoconstric-
tor response to a chronic inflammatory re-
sponse.
Asthma is the disease that has been most
strongly linked to production of cysteinyl leu-
kotrienes (105). This statement is supported
by (1)the increased levels of LTE, found in the
urine of asthmatic patients during an attack
and (2) the efficacy of leukotriene agents in
the disease. Urinary LTE, can be used as a
predictorlindicator for nocturnal asthma
(106). Other diseases in which elevated con-
centrations of the peptide leukotrienes have
been found include rhinitis, urticaria, arthri-
tis, ARDS, uveitis, and psoriasis (107).
5 AGENTS INHIBITING LEUKOTRIENE
BIOSYNTHESIS
The generation of leukotrienes depends on the
formation of LTA,, the key intermediate lead-
ing to either LTB, or the peptidoleukotrienes.
Thus, the most versatile and thus most widely
studied approach to the inhibition of leukotri-
ene biosynthesis involves the inhibition of for-
mation of LTA,, although attempts to inhibit
the enzymes involved in the transformation of
LTA, also offer interesting alternatives (LTA,
hydrolase, LTC, synthase), which are dis-
cussed later.
It is clear that 5-lipoxygenase (5-LO) [EC
1.13.11.341, through a two-step process, is the
enzyme responsible for the production of
LTA,. Thus the mechanism of action and, to a
lesser extent, the structure of 5-LO have
largely influenced the design and discovery of
inhibitors of leukotriene biosynthesis. This
enzyme has been the subject of intense re-
search since its discovery, as discussed below
(Section 5.1), and many inhibitors have been
described.
5-Lipoxygenase-activating protein (FLAP)
is also involved in the process by which AA is
converted to LTA, and a number of interest-
ing inhibitors have been described (Section
5.2).
The advantage of inhibiting 5-LO (or
FLAP) is essentially to block the effects of all
LTs and hence provide agents with therapeu-
tic potential in a wide range of allergic and
inflammatory diseases including asthma,
5 Agents Inhibiting Leukotriene Biosynthesis
COPD, fibrotic lung disease, allergic rhinitis,
rheumatoid arthritis, inflammatory bowel dis-
ease, and possibly psoriasis. Furthermore,
concerns over the likely heterogeneity of dif-
ferent functions and signaling pathways of LT
receptors and agonist-ligand specificities do
not affect this approach. The potential disad-
vantages arise from the possibility that com-
plete LT inhibition could give rise to undesir-
able side effects, although no indications of
any essential physiological role of LTs have
appeared since the discovery of these media-
tors. Indeed, recent work with 5-LO(-/-)-de-
ficient mice, generated by inactivation of the
5-LO gene (108, log), showed that these ani-
mals developed normally with no adverse
health effects, which suggests that 5-LO
blockade should not cause untoward effects.
In addition, studies on Zyflo (see next section)
indicate that inhibition of whole body leuko-
trienes at least to >80% would seem to be
broadly safe.
The endeavors to discover such inhibitors
and the successes to date are described in the
following sections.
5.1 Inhibitors of 5-Lipoxygenase ( 5 4 0 )
The mechanism of 5-LO appears to involve the
oxidized active Fe3+ state and the generation
of radical species
- (2). Known mechanisms of
hibition constitute trapping of radical inter-
ediates, ferric iron ligation, reduction of the
nheme iron, reversible binding at active or
her regulatory sites, and combinations of
ese effects in the same molecule. Conse-
ently, three broad classes of direct 5-LO in-
itors have evolved over the last 15 years:
redox," iron ligand, and "nonredox" inhibi-
rs. First, we will summarize the properties
of 5-LO in the light of these different methods
of inhibiting this enzyme.
5.1.1 Structure and Mechanism of 5-10. It
ould be noted that most of the original drug
scovery effort targeting 5-LO inhibitors was
ne in the absence of any detailed structural
owledge of the enzyme. The amino acid se-
uence of human 5-LO was not described until
985 (1101,when isolation and cloning showed
th it and rat 5-LO to be 78-kDa proteins
h 93%homology. In its active form 5-LO is
embrane bound; purification, therefore,
modifies its natural behavior. In 1991 recom-
binant methods allowed the preparation of
pure enzyme in milligram quantities (110)and
allowed the experiments that definitively
showed nonheme iron associated with highly
active enzyme obtained from a baculovirus ex-
pression system (111).
Interestingly, even at that time, a series of
five histidine residues was postulated to con-
stitute the potential iron-binding site, in that
they are also conserved in other lipoxygen-
ases, although site-specific mutagenesis stud-
ies indicated that three of these could be mu-
tated individually in 5-LO without loss of
either oxygenase or LTA, synthetase activity
(112,113). This proposal has now been refined
and it is believed that the nonheme iron is
bound permanently by His372, and
Ile673and that a fourth exchangeable ligand,
His367,is replaced by a reaction intermediate
during the catalytic cycle (114).This exchange
ligation is interesting from the point of view of
designing inhibitors because it suggests that
compounds in the appropriate oxidation state
with affinity for Fe3+ could also displace this
ligand and thus inhibit catalysis. Indeed, hy-
droxamate and N-hydroxyurea inhibitors may
possibly act in this way. Except for other li-
poxygenases, 5-LO shows little homology to
other proteins. The enzyme has recently been
shown to have a SH, domain that binds to the
growth factor bound receptor protein (115).
This interaction may be the site that allows
membrane association of the enzyme, al-
though that relationship has not been defini-
tively shown. Isolated 5-LO requires Ca2+,
ATP, and phosphatidyl choline for optimal ac-
tivity but its amino acid sequence has not been
found to show any strong homology with other
proteins possessing either Ca2+- or ATP-bind-
ing sites.
' Detailed structural information is still un-
available for 5-LO and thus has not been help-
ful in the design of 5-LO inhibitors. Initial re-
search attempts were also made difficult by
the complex kinetic behavior of this enzyme
(116-118).
Elucidation of the actual mechanism of
5-LO has been a difficult and often disputed
area. Here, we highlight the essential steps to
help clarify the inhibitory mechanism of the
compounds described hereafter. After activa-
 COX-2 Inhibitors and Leukotriene Modulators

tion (Fez+-Fe3+)during the lag phase, Fe3+

specifically oxidizes the diene of AA at position
5, to generate a pentadienyl radical cation. A
basic amino acid residue in the enzyme-active
site abstracts a proton and the ferrous iron is
reduced to Fez+.The pentadiene radical then
stereospecifically reacts with oxygen at C,,
forming a 5(S)-hydroperoxy radical that un-
dergoes electron transfer and reprotonation to
form 5-HPETE. Fe3+ is regenerated for recy-
cling. 5-HPETE is subsequently converted to
LTA, through deprotonation at C,, and epox-
ide cyclization. The participation of radical gle et al. (121). Although a large number of
species has been demonstrated by Mossbauer inhibitors have been examined in vitro and in
experiments (119), consistent with pentadi- vivo (21, no favorable clinical data have been
enyl radicals being intermediates in the cata- forthcoming from this approach.
lytic reaction.
From this reaction mechanism, as men- 5.1.3 Iron Ligands. Based on the hypothe-
tioned above, it is clear that many types of sis that incorporation of functional groups
molecules could inhibit 5-LO in vitro, espe- that could chelate to iron would inhibit 5-LO,
cially those with antioxidant properties. In- Corey (122) synthesized arachidonohydrox-
deed, several hundred patent applications amic acid and a number of N-alkyl derivatives
have been filed by over 40 different pharma- (3).These indeed proved to be potent 5-LO
ceutical companies since work has begun on
5-LO, claiming inhibitors with various de-
grees of potency and selectivity.

5.1.2 Antioxidants. 5-LO is particularly

susceptible to inhibition by compounds with
low redox potentials. Phenidone and BW-755C
(1,2, respectively) (120) were among the first
inhibitors (IC,, = 0.03-0.1 a, RBL-1) and
rapidly, elsewhere, many hydroxamate inhib-
itors were prepared that possessed high in
vitro potency.
Work at Abbott identified hydroxamates,
such as compound (4), by modification and

(1) Phenidone

compounds to be identified and other com-

pounds in this class are based around the het-
eroatom-substituted pyrazole ring. They are
loosely termed redox inhibitors because they
may, directly or indirectly, reduce either Fe3+
in the active enzyme or one of the radical in- simplification of the lipophilic chain. These
termediates to give the inactive Fez+ form. compounds are very potent in vitro and are
Early redox inhibitors were reviewed by Swin- well absorbed; however, they are rapidly me-
5 Agents Inhibiting Leukotriene Biosynthesis
tabolized to inactive carboxylic acids and show
short half-lives and low in vivo potencies
(123). Modification of the orientation of at-
tachment of the hydroxamate group (e.g.,
A-63162,s) had little effect on in vitro activity,
but A-63162 showed significantly improved in
vivo activity (124, 125).
Parallel work at Wellcome revealed similar
trends. That group discovered BW-A4C (61, a
hydroxamate similar to A-63162 in structure,
which showed good in vitro potency (IC,, =
0.14 ,uM, human PMN) (126).
Despite promising oral activity in animal
models, BW-A4C had poor pharmacokinetics
in human volunteers. Extensive metabolism
of BW-A4C occurred in humans (127), result-
ing in the accumulation of high concentra-
tions of metabolites. Additional modifications
were required to avoid the metabolic problems
encountered with hydroxamates to convert
this class into orally active clinical candidates.
5.1.4 N-Hydroxyureas. The substitution of
the hydroxamate with N-hydroxyurea, first
one at Abbott, provided compounds that
ere quickly shown to have superior in vivo
ehavior in animal models and pharmacoki-
etics. From a study of several hundred com-
unds containing the N-hydroxyurea moiety,
o (zileuton; A-64077, 7) emerged as the
didate for clinical trials (128, 129). In this
mpound the lipophilic benzyloxyphenol
(7) Zyflo (zileuton, A-64077)
moiety of A-63162 was replaced by a benzo-
thiophene, although the important a-methyl
was retained, with the result that zileuton,
like A-63162, contains a stereogenic center.
Zileuton is a potent 5-LO inhibitor i n uitro
and shows approximately 120-fold selectivity
over COX-1. Interestingly the R and S enanti-
omers that constitute zileuton were very sim-
ilar in biochemical potency (1301, varying by a
factor of only 2-3. These data imply that the
a-methyl site binds to an area of the enzyme
with considerable flexibility and that specific
ligand interaction with iron through the N-
hydroxyurea occurs in both enantiomers. In
contrast, minor modifications of the benzo-
thiophene ring resulted in significant loss in
activity (131).
The mechanism of 5-LO inhibition by N-
hydroxyureas and hydroxamates is clearly
more complex than mere iron chelation. There
is no doubt that these compounds bind
strongly to iron in solution (K = 1012/molfor
BW-A4C) and that they are not powerful redox
compounds, having relatively high electrode
potentials (E0.2V for BW-B70C, 8, and zileu-
ton, 7). Mechanisms for the activity of the
compounds remain complex (132).
5.1.4.1 In Vivo Activities and Clinical Stud-
ies with N-Hydroxyurea Inhibitors. BW-B7OC
is efficacious in allergen-induced bronchocon-
striction and late-phase lung eosinophil accu-
mulation in sensitized guinea pigs (133). Un-
fortunately, it produced kidney lesions in the
rat, preventing clinical evaluation. Zileuton
(Zyflo), however, was approved for use in the

treatment of moderate to severe asthma in
1996 as Zyflo. Several summaries of the clini-
cal studies on Zyflo have since been published
(134, 135). Thus we will summarize only the
pertinent development events and key clinical
findings. Zileuton is an orally active inhibitor
of 5-LO in rats, dogs, monkeys, and humans
measured by ex vivo inhibition of LTB, pro-
duced by addition of calcium ionohore A23187
in blood. Preclinical studies with the com-
pound showed it capable of inhibiting leuko-
triene-dependent inflammation in mice (136)
and inflammatory cell influx in rats (137). It is
also effective in guinea pig and sheep models
of antigen-driven bronchoconstriction (138).
In phase I, volunteer studies showed that
the inhibition of ex vivo-stimulated LTB, in
blood correlated with plasma drug levels
(139). Pharmacokinetic studies in phase I vol-
unteers determined that the major route of
metabolism of zileuton is glucuronidation of
the N-hydroxyurea group followed by urinary
excretion, and the oral half-life was estimated
to be 3 h (140). With this relatively short half-
life it was necessary to administer 600 mg four
times a day (qid) to maintain ex uivo-stimu-
lated LTB, inhibition in human blood at ap-
proximately 80% returning to control levels
after removal of the drug. No untoward side
effects were observed.
Consequently, Zyflo was thoroughly evalu-
ated in a number of clinical trials for effects on
models of asthma and in the treatment of
chronic asthma. Challenge models of asthma
with a variety of stimuli (allergen, exercise,
cold dry air, or aspirin) were successful. Re-
sults in aspirin-induced asthma were dramat-
ically effective, indicating that leukotrienes
are the primary mediators of this response.
Zyflo also shows remarkable anti-inflamma-
tory effects, as predicted from animal studies
(137). Eosinophil influx and albumin leakage
were reduced and the urinary increase in
LTE, was blocked (86%) (141, 142).
Zyflo was also found to be effective in
chronic asthma, as evidenced by two large pla-
cebo-controlled trials (143, 144). Improved
FEVl and reduced bronchodilator use were
the primary clinical endpoints. Interestingly,
Zyflo appeared to be most effective in moder-
ate to severe patients. Additional trials indi-
cated that Zyflo is also steroid sparing (145).
COX-2 Inhibitors and Leukotriene Modulators
5.1 A.2 Second-Generation N-Hydroxyureas.
During these trials it was realized that the
short half-life (3 h) and high clinically effective
daily dose of Zyflo (2400 mg) needed to be im-
proved. At Abbott, the search for more potent,
longer-acting N-hydroxyurea inhibitors fo-
cused on reducing the glucuronidation rate of
the N-hydroxyurea function. Various in vitro
models were used, particularly microsomal
preparations from human or monkey liver, to
assess new inhibitors (146). In this way, struc-
ture-activity relationship (SAR) studies indi-
cated that the lipophilic heteroaryl template
and the linking group to the N-hydroxyurea
could be profitably modified (131). Introduc-
tion of an unsaturated linking group was
shown to modulate inhibition and metabo-
lism. The spatial orientation of the lipophilic
template with respect to the pharmacophore
had a dramatic effect on pharmacological
properties. Acetylene linking groups signifi-
cantly decreased the rate of glucuronidation
(GT) (2, 139).
Inhibition of leukotriene formation in hu-
man whole blood was also improved. There
was little difference in in vitro activities be-
tween A-78773 and its enantiomers (9); how-
ever, the glucuronidation rate in monkey mi-
crosomes was lower for R(+) A-79175. The
relative rate correlated with in vivo elimina-
tion half-lives in cynomologus monkeys [4.7 h
for the racemate, 1.8 h for S ( - ) A-79176 and
9.0 h for R(+) A-791751, prompting a Phase I
clinical study in which R(+) A-79175 exhib-
ited an apparent half-life of 6.5 h for a single
600 mg p.0. dose, twice that of Zyflo (147,148).
Further chemical modification led to R(+)
ABT-761 (101,which was found to be even
more resistant to in vitro glucuronidation
(149). Its elimination half-life in monkeys was
16 h and consequent phase I studies in hu-
5 Agents Inhibiting Leukotriene Biosynthesis
(10) ABT-761
mans showed excellent bioavailability and
extended duration of plasma levels. Signifi-
cantly, a single 200 mg dose provided >95%
inhibition of ex vivo-stimulated LTB, for-
mation for up to 18 h. ABT-761 showed sig-
nificant protection against exercise and ade-
nosine-induced bronchoconstriction in asth-
matics (150, 151). However, idiosyncratic
liver toxicology precluded the further devel-
opment of this potent, effective 5-LO inhib-
5.1.5 Substrate Inhibitors. A number of
nhibitors just described. At Zeneca, this work
was begun in the late 1980s where "active-
site-directed" inhibitors were designed to fit
5-LO selectively without the need for redox or
strong iron ligand properties. This was partic-
ularly difficult in the absence of structural
dataon 5-LO; however, based on the hypothet-
ical mechanism discussed in Section 5.1.1,
which implies the presence of an Fe3+ atom
and a basic group adjacent to a lipophilic
pocket in the active site, lipophilic imidazoles
were initially synthesized to interact with
It was discovered that replacement of the
imidazole by a thiazole and introduction of
confonnational restriction with rnethoxy and
TB, inhibition) that importantly showed no
(11) ZM211965
COX-1 inhibition up to 100 pM in human
blood (152, 153). Cyclic voltammetry and iron
chelation measurements confirmed that this
(methoxyalky1)thiazoleseries is free from re-
dox and iron-complexing properties. The se-
ries differs from redox and the N-hydroxyurea
series in that it demonstrates enantioselective
inhibition of 5-LO both in vitro and in vivo
(154). This was the first evidence of 5-LO in-
hibitors forming enantiospecific interactions
with the enzyme. Thus, unlike the a-methyl-
ene center in the Abbott N-hydroxyurea se-
ries, the stereoselectivity of these ligands indi-
cates a close contact of the stereogenic center
and active site of the protein.
A number of steps were required to dis-
cover an orally bioavailable inhibitor in vivo
similar to that seen with the N-hydroxyureas
(2). The compound discovered was improved
by structural modification of the naphthyl
ring, and further introduction of a fluoro sub-
stituent to reduce susceptibility to metabo-
lism. This compound was ZD2138 (12) (IC,, =
0.02 pM, human whole blood) (155). ZD2138
possesses no redox properties and extensive
SAR data and detailed c~nformationalanaly-
ses suggest that the (methoxyalky1)thiazole
and tetrahydropyran (THP) series are related
(156). ZD2138 shows no inhibition of CO, even
up to 20,000 times the levels that inhibit 5-LO
in dog or human blood. ZD2138 does not an-
tagonize FLAP.
In phase I studies, ZD2138 was well toler-
ated up to 1000 mg per volunteer. A single oral
350 mg dose completely inhibited LT synthe-
sis ex vivo in stimulated blood for over 24 hand
the half-life in humans was estimated to be
12-16 h (157). Phase I1 clinical trials demon-
strated that in aspirin-sensitive asthmatic pa-
 COX-2 Inhibitors and Leukotriene Modulators

tients, 350 mg p.0. given 4 h before challenge

caused bronchodilation and inhibited the fall
in FEVl (158). However, in allergen-chal-
lenged asthmatics the same treatment with
ZD2138 had no effect on the early or late asth-
matic response (159). Further development of
ZD2138 was suspended (160). A detailed de-
scription of the discovery of ZD2138 can be
found in Ref. 161.
Extension of the work in the Zeneca group
around this series led to ZD4407 (13),which is

series of indole-3-alkanoic acids related to

extremely potent (IC,, = 0.02 a, human
whole blood leukotriene inhibition) and is de-
void of any autoinduction of liver enzymes
that had been observed preclinically for earlier
members of this series (162).
The methoxytetrahydropyran moiety has
been exploited by other groups. Merck Frosst
identified a class of lignans derived from a nat-
ural product, Justicidin E, as moderately po-
tent redox inhibitors of 5-LO (163). Abbott
also described compounds in this arena (164).
These compounds were very weak inhibitors
of 5-LO in broken cell preparations, as was
ZD2138, although many compounds with po-
tent whole blood activity were obtained. These
data imply a different mode of action for this
series than direct 5-LO inhibition and are in-
consistent with a substrate inhibitor.
5.2 Inhibitors of 5-Lipoxygenase-Activating
Protein (FLAP)
FLAP inhibitors are a novel class of com-
pounds that inhibit cellular leukotriene bio-
synthesis without acting directly on 5-LO. In-
stead, they bind to the 18-kDa membrane
FLAP, thus preventing the activation of 5-LO.
Research at Merck Frosst in 1989 (165,
166) led to the initial FLAP inhibitor, MK0886
(14), and consequently to the discovery of
FLAP itself. MK0886 was derived from a
indomethacin, which possessed the dual
pharmacological properties of TXA, recep-
tor antagonism and leukotriene biosynthe-
sis inhibition in intact cells.
These properties could be separated and
MK0886 had no effect on isolated, purified
5-LO, although in intact leukocyte prepara-
tions it inhibited LTs at low nM IC,, values
(3-5 nM)(167).Thus, a novel mode of action of
leukotriene inhibition had been discovered,
which was then exploited to find new inhibi-
tors. Indeed, it further helped the understand-
ing of the mechanism of 5-LO activation.
As discussed earlier, FLAP is associated
with a cellular membrane; initially, this was
believed to be the plasma membrane, but work
at Merck Frosst and Michigan (168, 169)
showed that localization is cell and activation
state dependent. In the neutrophil, inactive
cytoplasmic 5-LO translocates to membranes
on activation by Ca2+ and associates with
FLAP, as demonstrated in an elegant series of
experiments by two laboratories (170). The
nature of this association was initially thought
to consist of 5-LO possibly docking onto the
membrane-bound FLAP (171),although it has
been proposed that FLAP presents AA to 5-LO
in a conformation favorable for reaction (172).
Whatever the true mechanism, it is clear from
transfection experiments that the association
of 5-LO and FLAP is essential for intact cell
LT biosynthesis (173). The FLAP inhibitors,
like MK0886, inhibit complex formation, pre-
venting the complete translocation of 5-LO,
and block LT biosynthesis.
5 Agents inhibiting Leukotriene Biosynthesis
MK0886 was shown to have a high binding
af'6nity for FLAP (IC,, = 23 nM) (174). Clini-
studies with MK0886 in atopic asthmatics
(44%)asthmatic responses in pulmonary
dion to an allergen challenge, but only
owed modest inhibition of ex vivo iono-
esis and urinary LTE, excretion (175).
A key observation that would impact the
ign of new FLAP inhibitors was that an
er leukotriene inhibitor, Rev-5901 (15),
(15) Rev-5901
ch contains a quinolinylmethyloxy sub-
d protein. The quinoline moiety is a prom-
ent binding motif of many FLAP inhibitors.
wo FLAP inhibitors developed from these
ads are L-674,636 (16) and MK0591 (17).
Clinical studies with MK0591 were initially
of MKO59l (24, 12, and 1.5 h before aller-
1, leading to reduced bronchoconstriction
early (79%) and late (39%) asthmatic re-
nses. Furthermore, in contrast to MK0886,
biosynthesis ex vivo in stimulated whole
d from these patients was totally inhib-
h after allergen challenge (176). The study
extended to several hundred moderate
(16) L.
provements in pulmonary function tests in-
cluding FEV, and morning and evening peak
expiratory flow. Similarly, rescue P-agonist
use was diminished. Adverse effects were no
different from placebo. However, the com-
pound was not taken into chronic asthma
studies.
Also using the Rev-5901 lead, a series of
FLAP inhibitors were developed in which the
hydroxyl group of Rev-5901 had been replaced
by a carboxyl and the n-pentyl alkyl chain had
been cyclized to an a-cycloalky substituent.
From this series BAY XI005 (18)was selected
for clinical development.
The cyclopentyl substituent was also ex-
panded to cyclohexyl and cycloheptyl. The cy-
cloalkyl substituent provides an important
lipophilic interaction, which makes a signifi-
cant contribution to the affinity. Similar sub-
stituents in MK0591 that may correspond
with this key lipohilic interaction include the
t-butylthiol group or the kchlorobenzyl sub-
stituent. Stereoselective effects were ob-
served, the R enantiomers displaying a
greater affinity for human FLAP. Modifica-
tion of cyclopentyl to a cycloheptyl alkyl sub-
stituent and modification of the acid to a

(18) BAY XI005
(19) BAY Y1015
methanesulfonylamide provided the backup
clinical candidate, BAY Y1015 (19).An inter-
esting modification that has been applied to
MK0591 had been the insertion of an oxime
moiety(179)to connect the terminal carboxy-
late to the indole skeleton.
Analog(20)(180)was similar in preclinical
leukotriene inhibition but somewhat superior
in monkey pharrnacokinetics to MK0591 (R.
Bell, unpublished observations, 1994). Re-
ported SAR trends for structures related to
COX-2 Inhibitors and Leukotriene Modulators
(20)and MK0591 are similar; thiazoyl and
pyridine were found to be bioisosteric to quin-
oline in both series(181,182).This oxime can
be inserted with two orientations: the oxy-
imino derivative, shown where the carboxy-
late is attached to the carbon terminus of the
carbon nitrogen double bond; or the oxime
ether orientation, where the acid is attached
to the oxime as an 0-alkylated-a-acetic acid
oxime ether (179).The oxime moiety provides
a structural unit that can present the carbox-
ylate in different spatial orientations for bind-
ing. In addition the heteroatoms of the oxime
can possibly increase affinity through hydro-
gen-bonding interactions. Another potential
advantage of the oxime may be in improving
pharmacokinetic parameters through solva-
tion of the heteroatoms of the oxime; this sol-
vation can affect solubility and absorption.
The oxyimino orientation proved to be su-
perior. This modification has also been applied
to the Bayer series as shown (21).The S iso-
mer (183)was found to be the most potent
with the oxime modification (184). A draw-
back of the BAY X1005 and related candidates
such as(21)are issues of synthetic complexity
and resulting cost of goods associated with a
resolution or asymmetric synthesis. The key
elements of BAY X1005,the quniolylmethoxy,
the cycloalkyl, and the acid functionality, are
attached to a single carbon center. This stereo-
genic center could be eliminated if a second
quinolylmethyloxyphenyl substituent could
replace the lipophilic cycloalkyl substituent
but maintain the critical lipophilic interaction
5 Agents Inhibiting Leukotriene Biosynthesis
with the protein. This hypothesis was verified
by the symmetrical analog (23).
Development of this symmetrical core led
d without the oxime linker
e found with superior hu-
neutrophil potency and in vivo efficacy
ting LT formation in the rat.
is series culminated in the identification
This compound can be
by simple bisalkylation of
pensive 4,4-bis(hydroxy-
eny1)valeric acid, also known as diphenolic
d. Of the FLAP compounds synthesized at
bott, ABT-080 had superior activity in in-
biting rodent leukotriene formation, pulmo-
tigen-challenged guinea
cs in cynomolgus
VML530) has pro-
gressed to Phase I trials.
5.3 Inhibition of LTA, Hydrolase
chanism of action
[EC 3.3.2.6.1 is el-
a 70-kDa zinc-containing monomeric
R1
(23) H
(24) CH3
enzyme (186), which catalyzes the vinylogous
hydrolysis of LTA, to LTB,. The zinc ion is
essential for its hydrolase activity and this en-
zyme also exhibits aminopeptidase activity.
Indeed, it appears that the peptidase and ep-
oxide hydrolase activities occur at the same
active site (187). Exploitation of the amino-
peptidase function of LTA, hydrolase allowed
Samuelsson et al. (188-190) to produce a se-
ries of tight-binding inhibitors that also in-
hibit its epoxide hydrolase activity. Elabora-
tion of this type of inhibitor could deliver
highly potent and selective anti-inflammatory
drugs.
Further work has been published on devel-
oping a series of compounds based on the
p-mercaptoamino derivative, of which the R
enantiomer is two orders of magnitude more
potent than the S isomer (191).Acetylation of
the thiol or its conversion to thio ethers or a
hydroxy group results in substantial loss in
binding, thus suggesting that selectivity for
the free thiol arises from the formation of a
highly favorable zinc-sulfide interaction in the
enzyme-inhibitor complex.
The research on inhibitors of LTA4 hydro-
lase has recently been reviewed (192). Exten-
sive research at Searle provided for the only
LTA, hydrolase inhibitor taken to clinical tri-
als thus far. Early inhibitors described by that
group were based on a zinc-binding hydroxam-
ate lead, kelatorphan (25) (193). Unfortu-
nately, reversed hydroxamate compounds
with superior in vivo pharmacokinetics were
considerably less potent. This was also true of
carboxylic acids. Another series described by
the Searle group used SC-22716 (26) as anon-
zinc-chelating phenoxyethyl amino lead. This
compound had excellent potency against the

(25) kelatorphan
enzyme and in human whole blood LTB, inhi-
bition, but was poorly active in vivo (192).Fur-
ther SAR work around this series led to SC-
57416A (27) (194).This compound was potent
in rodent models of LTB, formation and had
excellent pharmacokinetics in several species
(195). In a preclinical efficacy trial in cotton-
topped tamarins this compound also had ex-
cellent activity in this animal model of human
colitis (196). However, "accumulation of a
long-lived metabolite and additional toxicity
issues precluded the compound from further
development" (197).
At this time there are no known LTA4 hy-
drolase inhibitors in development. Recent
studies in LTA, hydrolase-deficient animals,
however, continue to affirm the potential for
safe, effective inhibitors of this class (198).
COX-2 Inhibitors and Leukotriene Modulators
5.4 LTC, Synthase
LTC, synthase is an 18-kDa integral mem-
brane protein that catalyzes the conjugation
of reduced glutathione (GSH) with the epoxide
LTA, to form LTC,, the intracellular parent of
the cysteinyl leukotrienes. The purification
and characterization of LTC, synthase has
been hindered because of its instability in the
semipurified state and the lack of an abundant
source of this enzyme. In 1992 Nicholson (199)
was able to specifically label this 18-kDa
polypeptide in a human monocytic leukemia
cell line by use of photoaffinity probes and
showed it to be distinct from glutathione S-
transferase. Most recently, molecular cloning
of the gene for human LTC, synthase indi-
cates that this enzyme shows significant ho-
mology to FLAP (Section 5.2) (200, 201). De-
tails of the biochemistry of this enzyme have
been recently reviewed (201, 202).
Perhaps the most important information
leading to inhibitors comes from the fact that
LTC, synthase has significant homology to
FLAP (200). An overall homology of 31% is
seen for these two proteins with 44% in the
N-terminal two-thirds of the proteins. FLAP
lacks the amino acids thought to be used in
catalysis by LTC, synthase. Early studies in-
dicated that FLAP inhibitors also inhibit
LTC, synthase, albeit in the f l range (200,
201). Surprisingly, little medicinal chemistry
has come forth in recent years describing new,
more potent inhibitors.
6 AGENTS ANTAGONIZING
LEUKOTRIENES
6.1 Peptide-Leukotriene Antagonists
6.1.1 Introduction. There have been thou-
sands of compounds prepared as CysLT antag-
onists, of which perhaps two dozen have made
it to Phase I safety studies in humans. In this
section, we focus on a few key compounds, es-
pecially those that have advanced to clinical
studies. For more detail, the reader is referred
to several excellent reviews by Shaw (2031,
Salmon (204), and Brooks (205).
Testing for leukotriene antagonism as for
LT inhibition has required many biochemical,
pharmacological, and clinical assays. Because
6 Agents Antagonizing Leukotrienes

of the variety of assays, only a few that are screening of a compound collection or varia-
critical for understanding this section are de- tion of one of the two known ligands, FPL-
scribed. Early researchers tended to measure 55712 or the leukotrienes.
inhibition of the contraction of a tissue sample FPL-55712 (28) was developed by chemists
(primarily guinea pig trachea or ileum) in- at Fisons Pharmaceuticals working from a
duced by a standardized unit of "SRS-A" that
had been isolated and (partially) purified from
a biological source. Later, after synthetic
(pure) leukotrienes became available, similar
assays putting them into service became
widely used (e.g., determination of the per-
centage inhibition of the 8 nM LTD,-induced
contraction of guinea pig tracheal spirals at a
specific drug concentration). For compounds
warranting greater effort, either IC,, values
or the calculated pKp (negative log molar dis-
sociation constants) values for inhibition of
the LTD,- and LTE,-induced contraction of (28) FPL 55712
specific tissues (e.g., guinea pig tracheal or il-
eal strips, or human bronchus) were deter- lead that they had discovered by broad screen-
mined. In some cases the pA, values (the af- ing of a set of antiallergic compounds against
ty of an antagonist drug as generally an SRS-A-based assay. This was accomplished
etermined from the mean of equally effective before the structure of SRS-A had been as-
onist doselresponse ratios in the absence or signed as a mixture of the cysteinyl leukotri-
resence of a fixed concentration of antago- enes. FPL-55712 served not only as a starting
st) against LTD, or LTE, were calculated. point for other chemists' efforts to develop
or reasons that are not clear, most antago- specific CysLTl antagonists but also helped in
nists tend to profile as being more potent the biological isolation and structure elucida-
against LTD, on guinea pig ileum than on tion of the LTs.
guinea pig trachea. For comparison purposes,
he IC,,, pKp, and pA, values in this section 6.1.2 FPL-55712-Derived Compounds
ere determined on guinea pig trachea, unless 6.1.2.1 L Y171883 (Tomelukast). The most
herwise indicated. significant of the early CysLTl antagonists
Later, LTD,-binding assays measuring dis- that progressed to clinical evaluation was
cement of [ 3H]LTD, from guinea pig lung LY171883 (29, tomelukast). Using explora-
mbranes were developed. Subsequently, a
inding assay using the human receptor in
937 cell membranes has been used by some
ups. Study of the binding assay revealed
at guinea pig tracheal receptors could be
ubdivided into high and low affinity classes,
h LTE, preferentially acting at the high
nity site (206). Comparison of the func-
ional results with several compounds showed
hat the receptor in human bronchi most
losely resembled the guinea pig LTE, recep-

In the absence of a structure or sequence (29) tomelukast (LY 171883)

r the CysLTl receptor, the starting points
r developing antagonists were limited. The tion of the SAR around FPL-55712, the group
hoices were either a novel lead obtained by at Eli Lilly & Co. first simplified the carboxy-

substituted chromanone, resulting in an ana-
log containing an aliphatic acid chain. They
then made the key discovery that replacement
of the terminal carboxylic acid group with the
bioisosteric tetrazole functionality afforded a
significantly improved profile both in vitro and
in uiuo (208). Optimization of the chain be-
tween the tetrazole group and the hydroxyace-
tophenone group yielded LY171883.
In anesthetized guinea pigs LY171883 3-30
mg/kg p.0. given 2 h before challenge dose-
dependently inhibited the increase in pulmo-
nary resistance induced by i.v. LTD, (209). In
conscious guinea pigs, aerosolized LY171883
was able to reverse the bronchoconstriction
induced by either LTD, or LTE,. Clinical tri-
als with LY171883 were pursued to a Phase I1
trial in chronic asthma that, although show-
ing small significant effects on lung function,
was not deemed potent enough to proceed.
Merck Frosst also worked in this area but did
not find clinical candidates (2).
6.1.3 Leukotriene Analog Antagonists
6.1.3.1 SKF 104353 (Pobilukast) and SKF
106203. SKF 104353 (30) (210) and SKF
106203 (31)(211) were both developed by the
SK&Fgroup through the sequential modifica-
(30) SKF 104353 (pobilukast)
(31) SKF 106203
COX-2 Inhibitors and Leukotriene Modulators
tion of the structure of LTD, and were the first
CysLT1antagonist clinical candidates to be so
derived (212).
Although SKF 104353 is about 10-fold
more potent in vitro than SKF 106203, it has
very poor oral bioavailabilitysuch that its clin-
ical development was limited to aerosol formu-
lations. In contrast, SKF 106203 had much
better oral bioavailability and entered early
clinical studies as an oral formulation. SKF
104353 is a potent, competitive inhibitor of
C3H]LTD4,binding to both human and guinea
pig lung membranes with Ki values of 12 and 5
nM,respectively (213). In functional assays it
inhibited the actions of LTD, on human bron-
chus (pA, = 8.2) and guinea pig trachea (pA,
= 8.6) (214).Similarly, in functional assays on
these two tissues, using LTE, as the agonist, it
afforded pKp values of 8.9 and 8.2, respec-
tively. SmithKline had some early clinical suc-
cess with SKF 104353 but not sufficient to go
to pivotal trials (205).
6.1.3.2 L Y170680 (Sulukast). Concurrent
with the efforts on FPL-55712 analogs at Eli
Lilly's U.S. labs, a second team at their UK
labs developed LY170680 (321, starting from
(32) LY 170680 (sulukast)
the structure of the natural agonists (215).
This compound was also dropped from devel-
opment after some antigen-challenge trials
where the drug was given as an aerosol.
6.1.3.3 Bayer x7195. The sequence of
steps taken by the Bayer group in going from
leukotriene D, to Bayer x7195 (33) has been
described in two articles (216,217). This inter-
esting candidate incorporates several of the
discoveries in LT analogs published earlier by
other laboratories. For example, optimization
of activity, particularly oral activity, required
both shortening the linking group to the C,-
carboxylic acid and removal of the hydroxyl
substituent, both precedented by the efforts at
 Agents Antagonizing Leukotrienes
(33) Bayer x7195
&F laboratories, that led to SKI? 104353
d SKF 106203. Use of thepara-alkoxy-sub-
ituted homo-cinnamyl replacement for the
ene backbone was also first described by the
ca team (218).
Bayer x7195 inhibits the binding of
i3H1LTD, to guinea pig lung membranes with
a pKi of 7.8 (219). In functional assays it had
values of 8.4 and 8.2 against LTD4-in-
ced contraction in guinea pig trachea and
man bronchi, respectively. In vitro, about 1
Bayer x7195 was also effective at blocking
antigen-induced contractions of trachea
om sensitized guinea pigs and the anti-IgE
sponse of human bronchi. In the anesthe-
zed guinea pig, orally administered Bayer
C1
(34) montelukast sodium (MK-0476, Singular)
C02H
--
-
S
'0- COzH
x7195 blocked LTD4-induced bronchocon-
striction (ID,, = 3 mglkg) for at least 8 h.
Topically administered Bayer x7195 was also
effective in the guinea pig model, whether
given by aerosol (ID,, - 200 ng) or as a powder
(ID,, - 30 pg). Again, although the compound
gave significant improvement in FEV, in
Phase I1 trials in chronic asthma, the com-
pound was not pursued to pivotal trials.
6.1.3.4 MK-0476 (Singulair, Montelukast
Sodium, 34) and Its Precursors. Because the
series of compounds exemplified by MK-0571,
MK-0679, and MK-0476 (34-36) contain mi-
metics for the three regions of LTD, (lipid,
acid, peptide) they may be viewed as leukotri-
ene analog antagonists. However, unlike the

(36) MK-0679 (verlukast)
other antagonists in this section, the starting
point for these compounds was not the struc-
ture of the leukotrienes. Instead it was a styryl
quinoline-containing compound (37) that was
found, by broad screening, to be a weak LTD,
antagonist. This lead appears related to a se-
ries of quinoline-containing LTD, antagonists
(2201, derived from REV 5901, that are no
longer in development and that are not cov-
ered in this review.
The chemists at Merck hypothesized that
compound (37) was mimicking the conjugated
olefin (lipid) region of LTD, and chose to add
mimetics for the acid and peptide regions
through the use of a dicarboxy-dithioacetal de-
rivative first pioneered by the SK&F group.
This change afforded a compound that dis-
played a tremendous increase in in vitro po-
tency. However, the dicarboxy group appeared
to be detrimental to oral efficacy. Replacement
of one of the carboxyl groups with an N,N-
dimethylamide improved oral efficacy and
yielded MK-0571 (35) (221), which entered
clinical development. This racemic compound
was found to be a potent inducer of liver per-
oxisomal enzymes and was replaced in the
clinic by its (R)-enantiomer MK-0679 (ver-
lukast, 36) that had similar LT antagonist ac-
tivity but an improved safety profile.
In extended studies this compound also in-
duced (limited) liver function abnormalities in
animals and in the clinic. An extensive effort
by the Merck group to find a compound in this
COX-2 Inhibitors and Leukotriene Modulators
series that was devoid of peroxisomal enzyme
induction led to the discovery of MK-0476 (34,
montelukast, Singulair) (222).
Montelukast is significantly more potent
than either MK-571 or MK-0679 in vivo. An
i.v. dose of 10 pg/ kg MK-0476 produced a 70-
fold and a greater than 100-fold shift in the
dose-response curves to i.v. LTD, in the anes-
thetized guinea pig, at 5 or 15 min pretreat-
ment times, respectively. The i.v. ED,, at
inhibiting the LTD,-induced bronchoconstric-
tion was estimated at 0.001 &kg. Monte-
lukast had a long functional half-life after i.v.
administration in the guinea pig. As discussed
below, montelukast has been successfully
tested in human asthma and approved for hu-
man use.
6.1.4 Peptide-Leukotriene Antagonists of
Diversified Structure
6.1.4.1 O N 0 1078 (Pranlukast). ON0 1078
(pranlukast, 38) is not a member of either the
FPL-55712- or the leukotriene D,-derived sets
of antagonists. It was developed from a weak
lead, compound (19) (IC,, = 14 p M versus
LTD, on guinea pig ileum). Replacement of
the benzoic acid group with a chromanone car-
boxylic acid (similar to that found in
FPL-55712) resulted in an analog that dis-
6 Agents Antagonizing Leukotrienes
(39) ICI 198,615
ed a 150-fold increase in potency in uitro
, = 100 nM) and modest levels of in vivo
ivity (ID,, = 500 p&g in the guinea pig
ilar to the discovery that led to
171883, replacement of the carboxylic acid
ole yielded increases in both in
and i n vivo potency (not illustrated). Op-
ation of the lipophilic alkyl tail then
ed ON0 RS-411, the hemihydrate form of
ch has been clinically developed as ON0
O N 0 1078 inhibited the binding of
HILTD,, on guinea pig lung membranes
6 nM (223). I n uitro, it had
values of 10.40 and 7.5 against LTD, on
pig ileum, with an IC,,
ue of 0.044 nM (224). I n uiuo, it was effec-
1.0 pgkg) against LTD,-induced
in guinea pig when ad-
istered i.v. 2 min before LTD, (225). ON0
against LTD, (5 ng), in-
in guinea pig was inhib-
d by ON0 1078 at a dose of 30 mgtkg p.0.
(40) zafirlukast (ICI 204,219, Accolate)
6.1 .4.2 ICI 198,615 and ICI 204,219 (Acco-
late, Zafirlukast). Several articles have re-
viewed the discovery of Zenaca's series of in-
dole-based CysLTl antagonists exemplified by
ICI 198,615 (39) and ICI 204,219 (40, Acco-
late, zafdukast) (227-229).These compounds
were developed from a novel hybridization be-
tween a leukotriene analog and a hydroxy-ace-
tophenone series of antagonists (230,231).
The first member of this series, ICI 198,615
(39) showed remarkable in uitro potency and
selectivity as a CysLTl antagonist when it
was disclosed. It inhibited the binding of
[3H]LTD4on guinea pig lung membranes with
a pK, value of 9.6 (232).On guinea gig trachea
respectively. ICI 198,615 was also a potent an-
tagonist of LTD, in human bronchi with a pK,
value of 9.2. It also showed i n uiuo antagonist
activity in guinea pig in both the classic anes-
thetized pulmonary mechanics model and in a
new conscious labored abdominal breathing
, showed poor oral bioavail-
198,615 was not developed
further. Later studies that have shown ICI
198,615 (i.m.) reduces antigen-induced airway
hyperresponsiveness in monkeys have con-
0

firmed its importance as a standard and indi-
cated yet another mode of action for such an-
tagonists (234).
This team introduced ICI 204,219 (40) as
its first broadly studied CysLTl antagonist
clinical candidate (235-237). In vitro, this
compound has an excellent profile that is sim-
ilar to that shown by ICI 198,615. In human
lung membranes it inhibited the binding of
L3H]ICI198,615 with a Ki value of 3.70 ? 2.90
nM (n = 5). The pKp values for inhibition of
the LTD,- and LTE4-induced contraction of
guinea pig tracheal strips were concentration
independent and were, respectively, 9.52 +
0.12 and 9.67 + 0.30, at 3 nMICI 204,219. The
distinguishing characteristic of ICI 204,219 is
its excellent in vivo profile. It had excellent
bioavailability in rat (68%)and dog (67%).The
overall profile of ICI 204,219 was such that its
oral formulation was chosen for extensive clin-
ical evaluation as Accolate (zafirlukast).
6.2 CysLTl Antagonist Clinical Studies
Inhalation of peptide leukotrienes has been
shown to induce a reproducible, robust, well-
tolerated bronchoconstriction in humans. Be-
cause of this, most clinical studies of CysLTl
antagonists have begun with an assessment of
their in vivo potency through measurement of
their ability to inhibit LT-induced broncho-
constriction. Because these studies can be
done over a broad time range, they can also be
used to determine human pharmacodynamic
values. The most frequent measure was the
-
shift in the LT-induced dose-response curve
that is produced by a specified dose of a drug at
a particular pretreatment time. Friedman
(238) has reviewed the pulmonary-oriented
clinical results obtained with most of the earlv"
peptide leukotriene antagonists and lipoxy-
genase inhibitors.
LY171883 was the first CysLTl antagonist
to be evaluated in broad clinical trials (239).It
was eventually withdrawn from clinical eval-
uation as a result of both long-term toxicity
seen in mice and induction of peroxisomal
liver enzyme in rats (240,241). In spite of this,
it was an especially important compound be-
cause it produced encouraging clinical results
that appeared to confirm the hypothesis link-
ing the cysteinyl leukotrienes to disease and
COX-2 Inhibitors and Leukotriene Modulators
thus acted as a strong spur to the development
of other CysLTl antagonists.
Aerosolized SKF 104353, 100 and 800 pg,
shifted LTD4-induced bronchoconstriction by
about 10-fold in normal and asthmatic individ-
uals, respectively, when administered 2-3.5 h
before challenge (242). The same dose pro-
tected against exercise-induced asthma (243).
SKF 106203 was examined in normal volun-
teers and a 200 mg p.0. dose was effective in
reducing LTD4-induced bronchoconstriction,
with the maximal effect being observed 8 h
after dosing (244).
ONON (pranlukast, ONO-1078, ONO-RS-
411 hemihydrate) was approved in Japan in
1995 and is the first CysLTl antagonist to be
commercially available. However, it has not
yet been approved in other countries.
The Merck group has entered the greatest
number of CysLTl antagonists into clinical
studies. Extensive trials were performed with
MK-0571 (L-660,711), its single enantiomer
MK-0679, and with MK-0476. This compound
montelukast (Singulair) has been approved
for clinical use in the United States and inter-
nationally for the treatment of asthma in
adults and children (245).
Montelukast underwent extensive clinical
testing. The studies used to support the clini-
cal use of this agent have been reviewed in
several venues (256-258). A daily oral dose of
Singulair controlled asthma symptoms in 3-
and 9-month extension trials. As with Zyflo
the use of Singulair also allowed steroid taper-
ing in moderate to severe asthmatics.
Zafrlukast formulated as Accolate was the
first leukotriene modulator approved in the
United States. This compound has also been
extensively reviewed (256-258). Given 40 mg
bid it has been shown to be clinically effective
in the treatment of asthma.
Since the registration of Accolate, Zyflo,
and Singulair (Table 5.21, most clinical studies
have centered on the effects of these agents on
inflammatory processes. The clinical trials
that formed the basis for their approval dem-
onstrated that these agents are safe and effec-
tive, and subsequent studies have also borne
this out (251,252). These agents are especially
effective in aspirin-sensitive asthma and exer-
cise-induced asthma. They are also effective in
treating the upper airway responses to IgE-
7 Leukotriene B, Antagonists
mediated immunological medial nasal conges-
tion, although none of the agents has regula-
tory approval for the nasal indication.
Yanase and David-Bajar have reported that
Singulair has a modest but significant effect in
treating atopic dermatitis (253). Asero (254)
has reported that in NSAID-induced uticaria
that Singulair may also have an effect. Acco-
late has also been reported to have an effect in
atopic dermatitis (255). In addition, Zyflo has
shown efficacy in significantly improving the
symptoms in a small open label study in atopic
dermatitis (256). There have been, however,
no large clinical trials to date to define the
potential activity of these agents in the treat-
ment of atopic dermatitis.
The major side effect that has been re-
ported for the CysLTl antagonists is Churg-
Strauss syndrome (257). A recent review of
the literature suggests that Churg-Strauss is
related to an underlying eosinophilic disorder
in patients in whom corticosteriods use was
being reduced, and thus not directly associ-
ated with the antagonists (257,258). Thus far,
this syndrome has not been reported with
zyflo.
7 LEUKOTRlENE B, ANTAGONISTS
7.1 Introduction
Although the structure of LTB, was discov-
ered at about the same time as the cysteinyl
leukotrienes, the pace of development of LTB,
antagonists has lagged significantly behind
that of CysLTl receptor antagonists. This de-
iay may be attributed to the fact that, unlike
with the CysLTs (SRS-A), there was not, at
first, a clear recognition of which pathophysi-
ologies to associate with LTB,. Also, as limited
as the starting points for drug development
were in the SRS-A area, they were even more
limited in the area of LTB, antagonists. Most
research teams began their efforts with the
structure of LTB,. Other teams started with
known LTD, antagonists and hoped that, be-
cause LTB, and LTD, were biosynthetically
related, some of these molecules would display
weak LTB, antagonist activity. Several previ-
wsreviews on the development of leukotriene
B4antagonistshave been published (259-261)
and, although clinical data on LTB, antago-
nists are much more limited than with the
LTD, antagonists, this section focuses on the
development of those compounds that have
advanced into clinical studies.
The in vitro and in vivo activities of LTB,
antagonists have been evaluated by use of sev-
eral different assays. Binding assays have
been developed that measure the inhibition of
binding of [ 3H]LTB4to guinea pig lung mem-
brane (262)and to isolated human neutrophils
(263). Functional activity has been assayed
through inhibition of the LTB,-induced con-
traction of guinea pig lung parenchyma (262)
and through the inhibition of the LTB4-in-
duced up-regulation of CDllbICD18 expres-
sion in human neutrophils. In vivo, in guinea
pigs or cynomolgus monkeys, inhibition of
LTB,-induced neutropenia and interdermal
neutrophil migration have been examined.
7.2 LTB, Antagonists Related to HAP-Type
LID, Antagonists
A team at Eli Lilly discovered that moving the
alkyl chain from the hydroxyacetophenone
(HAP) &position, as found in FPL-55712 (28),
resulted in selective LTB, antagonists. For ex-
ample, LY255283 (41) inhibited LTB, binding
to neutrophils with an IC,, value of 87 nM
(264). Further work in this class yielded
LY293111 (42), which was active in blocking
LTB,-induced bronchoconstriction in vivo
(265). A publication on LY293111 has ap-
peared describing its pharmacology in vivo
(266). Unfortunately, the compound was not
effective in clinical trials in asthmatics.
The Searle group apparently started with
FPL-5512 analogs and arrived at SC 53228
(43),which antagonized LTB, binding with an
 COX-2 Inhibitors and Leukotriene Modulators

IC,, value of 1.3 nM.The compound was also

active in blocking PMA-induced ear edema
(<2.5 mglkg).

7.3 LTB, Antagonists Derived from the

Structure of LTB,
A number of agents have been reported based
on the structure of LTB,. The early pharma-
cology was previously reviewed (2). The com-
pounds that have advanced furthest are
ONO-4057 (46) and two compounds from
SmithKline Beecham, SB 201993 (44) and SB
209247 (45). Early ON0 compounds used a
variety of groups to replace the triene in LTB,
(267). The diacid ONO-4057 (46) had an IC,,
in binding assays of 15 nM and was active in BIIL 284 (48) was longer acting in monkeys
the guinea pig in LTB,-induced neutropenia. than either CGS-25019c or LY 293111.
SB209247 was also orally active in rodents
7.4 Clinical Trials with LTB, Antagonists
(268) and was taken to clinical trials (2). CGS-
25019c (47), also taken to phase I trials, was Clinical success has not yet been achieved with
unique compared to earlier discussed com- LTB, antagonists. It is unclear whether this is
pounds in that it had an amidine group func- because LTB, is only one of many leukocyte
tionality instead of an acidic group (269). More chemoattractants in inflammation or whether
recently, a series of prodrugs of an amidine a significant portion of the biology of LTB, is
group were described by Boeringer Ingelheim directed by binding to BLTR2. Several com-
(270). The lead compound from this series, pounds have been in phase I trials (2) but no
11 8 Biochemistry and Structural Biology of Cyclooxygenases
t
I
lbstantial success has been reported and no
~mpoundshave been registered. Attempts in
asthma and psoriasis have been deemed mar-
ginal or unsuccessful (271-272).
8 BIOCHEMISTRY A N D STRUCTURAL
OLOCY OF CYCLOOXYCENASES
8.1 Prostaglandin Discovery and the
mnection to NSAlDs
.ostaglandins are the earliest of the eico-
noids to be discovered. Originally described
r von Euler in the late 1940s as the active
incipal components in semen that induced
erine contractility (273), the structures of
e most important of these (PGE,, PGF,,,
3D,) were elucidated in the 1960s by Berg-
rom, Hamberg, Samuelsson, and others
(47) CGS 25019C
N
0
F"
/
(48) BIIL 284
(Fig. 5.3) (274). The multiple activities of
these substances were the topic of intense re-
search in the 1960s and 1970s, where roles in
reproductive, cardiovascular, pulmonary and
renal physiology, and pathophysiology (pain,
inflammation, cancer) -were identified. The
discovery of thromboxane (275) and prostacy-
clin (276) came later and expanded the role of
the eicosanoids as powerful substances in hu-
man physiology.
 COX-2 Inhibitors and Leukotriene Modulators

Cell membrane

,
phospholipid pools

EH
Phospholipase(s)

Other /
lipoxygenases
5-Lipoxygenase

Other HETEs Arachidonic acid Leukotrienes

and diHETES HETEs
COX-1 OH
COX-2

-
OH
0'
PGE2 PGF2,
0
CH3
-
OH

PGH 2
HO"'
- CH3
OH -
OH
PGD2 PGI2

Figure 5.3. Cyclooxygenase pathway.

In a parallel fashion, albeit starting much
earlier, the discovery of nonsteroidal anti-in-
flammatory drugs (NSAIDs) were derived
from early folk medicine use of natural sub-
stances, the active agent of which was ulti-
mately discovered to be aspirin in the 1800s.
Once the negative gastric side effects of aspi-
rin were established around 1940, a number of
synthetic agents were developed with the goal
of achieving safer drugs. These drugs were
eventually classified as NSAIDs, in contrast to
steroids, the other major class of anti-inflam-
matories then available.
Assignment of NSAIDs as cyclooxygenase
inhibitors and thus prostaglandin inhibitors,
was made in the early 1970s by Vane and col-
leagues using classical tissue strip pharmacol-
ogy (277). This discovery was elegantly fol-
lowed by Majerus et d., who identified the
amino acid site of the covalent acylation of
platelet cyclooxygenase by aspirin (278).
8.2 Cyclooxygenase 1
Cyclooxygenase 1 (COX-1) is the common
name used to describe the protein prostaglan-
din H synthase 1 (PGHS-11, which performs
both peroxidase and cyclooxygenase reactions
to make PGH, the precursor to prostaglan-
dins E, D, F2, prostacyclin, and thromboxane
A, (see Fig. 5.3). Originally described as an
8 Biochemistry and Structural Biology of Cyclooxygenases
enzymatic activity in seminal vesicles, it was
purified in 1977 by two groups (279). The ac-
tive enzyme is a homo-dimer with subunits of
74,000 Da. As described earlier (2), the dimer
containing two noncovalent heme moieties
initially interacts with endogenous peroxide
where a two-electron reaction occurs. A one-
electron translocation provides the tryosyl
radical reauired for interaction with arachi-
donic acid. When the cyclooxygenase site is
occupied by arachidonic acid, the 13 pro-S hy-
drogen is extracted to yield an arachidonyl
radical. The fatty acid radical then reacts with
molecular oxygen to form the 11-hydroxylrad-
ical, which subsequently cyclizes a Cll to C,
endoperoxide moiety, interacts with a second
molecule of O,, and ultimately produces
PGG,. As described in more detail (280) the
tyrosyl radical can cycle through the peroxi-
dase site if no arachidonate is available. Al-
though reasonably well understood and ac-
cepted, there are still controversies in the
PGH mechanism (281). Subsequent to purifi-
cation efforts, cyclooxygenase 1 was cloned
first from sheep (282) and then from other spe-
-
cies. Ex~ression of COX-1 occurs in most if not
all mammalian tissues and cells in culture
(283, 284). The crystal structure of ovine
COX-1 was solved in 1994 and allowed rigor-
ous interpretation of numerous biochemical
and site-directed mutagenesis work (285). Im-
portant aspects of the crystal structure are
discussed below in comparison to COX-2.
8.3 Cyclooxygenase 2
As described above, the first described cyclo-
oxygenase was purified and characterized in
the late 1970s. This work -provided the molec-
ular framework for connecting the biochemis-
try of the enzyme studies predominantly in
seminal vesicle preparations and in the plate-
let, with the biology that had expanded to in-
clude all major organ systems and most cell
types. In the 198&, however, several groups
began to obtain data inconsistent with the
characteristics of the cyclooxygenase in plate-
lets and seminal vesicles. In particular, hor-
monal control of prostaglandins in cells such
as fibroblasts and macrophages (286,287) and
in vivo (288,289) were inconsistent with a sin-
gle enzyme. These publications proposed two
pools of cyclooxygenase, one static and unre-
231
sponsive to steroids and the other inducible
and inhibited by steroids such as dexametha-
sone. Some investigators also reported in-
creased enzyme concentrations in response to
growth factors by Western blot with antibod-
ies thought to be specific for COX-1 (287).
The discovery of a second cyclooxygenase
enzyme initiated an explosion of research.
This research showed that COX-2 was distin-
guished from COX-1 by a slightly larger mo-
lecular mass, rarity of mRNA expression, and
induction by growth factors and cytokines
(280). Further work has yielded information
with significant detail in the biochemical, cel-
lular, pharmacological, and structural differ-
ences between the two enzymes.
8.4 Structural Features of COX-1
and COX-2 Enzymes
A large body of structural biology information
on the two cyclooxygenases, COX-1 and
COX-2 (PGHS-1 and PGHS-2) now exists.
Overall, the data indicate a remarkable degree
of similarity between the enzymes. They differ
in length by only 11 amino acids, with COX-2
being slightly larger. The differences in se-
quence are predominantly in the membrane-
binding domain. The overall structure of the
human and mouse COX-2 is superimposable
on the ovine COX-1 structure (280). Both en- .
zymes are dimers with three structural do-
mains: an N-terminal EGF domain of approx-
imately 50 amino acids, a membrane-binding
domain of about 50 amino acids, and a large
C-terminal globular domain that contains the
catalytic domain. The dimer interacts with the
membrane in a novel way, associating with the
inner leaflet but not penetrating the entire
membrane, as supposed before the crystal
structure of COX-1 was elucidated (285).
The nature of the active site. how substrate
is bound, and in particular how inhibitors bind
in the two active sites have been extensively
studied (280, 290). As COX-2 was discovered,
the availability of the COX-1 crystal structure
allowed for models, particularly site-directed
mutagenesis, to explore differences in the two
active sites. Early modeling studies indicated
clearly that the first layer of amino acids in the
active site were largely conserved, with the
exception of valine509(291). A few differences
also occur at the next "layer" of amino acids

surrounding the active site. Good evidence for
the importance of the valine residue comes
from mutation of that residue causing changes
in the selectivity of agents (292)and from crys-
tal structures with SC-558 (51c), where it was
found that this inhibitor "wedges" the sulfon-
amide group into a hydrophobic side pocket
that is inaccessible in COX-1 (292). Movement
of the valine and insertion of sulfonamide or
methyl sulfone moieties common to many se-
lective COX-2 agents may be critical for the
selectivity seen for these agents. Structures
with inhibitors and using nitroxide-labeled
enzyme show clearly that the active site is
modified by inhibitor and substrate binding
(293). Indeed, structural flexibility may be im-
portant for catalysis (293). Recently, the crys-
tal structures of COX-2 with arachidonic acid
and PGG, (294) in the active site have been
determined. In particular, the structure with
PGG, is supportive of earlier biochemical and
modeling data, illustrating again the similari-
ties between the two enzymes.
8.5 Biosynthesis of Prostaglandins
Prostaglandins are made by nearly every or-
gan system, tissue, and cell in the body. Under
normal conditions this is predominantly
driven by COX-1. This enzyme is broadly ex-
pressed and is constitutively active (290). Un-
der homeostatic conditions, prostaglandin for-
mation is controlled by substrate availability
and perhaps peroxide tone (295). Most cells
and tissues tightly regulate free fatty acid con-
centrations to be very low. The chief enzymes
responsible for this are the fatty acid transac-
ylases, which rapidly reacylate free fatty acid
into either phospholipids or triglycerides.
Agents that stimulate production of prosta-
glandins from COX-1 have primarily been
agents that cause a calcium spike inside cells.
In contrast to COX-1, expression of COX-2 is
normally limited in the body to specific cells in
kidney, brain, and pancreas (290). In general,
COX-2 must be induced for substantial con-
centrations of prostaglandins to occur (290). A
plethora of inducers have been reported and
fall into several classes including cytokines,
growth factors, and hormones (296-298).
COX-2-dependent prostaglandin production
thus occurs over hours rather than minutes,
in contrast to COX-1-driven eicosanoid pro-
COX-2 Inhibitors and Leukotriene Modulators
duction that requires only seconds or a few
minutes. Additionally the expression of
COX-2 enzyme is usually the rate-limiting
step for COX-2-driven eicosanoid formation,
not substrate release.
Several groups have explored the molecu-
lar details of how COX-2 expression is in-
duced. It appears that agents that induce
COX-2 expression do so by both inducing
COX-2 mRNA production as well as stabiliz-
ing the mRNA. Recent work by Song et al.
indicates that COX-2 expression is normally
silenced through a hypermethylation mecha-
nism (299). Induction of COX-2 mRNA has
been reported to occur through IL-1 media-
tion, ceramide-dependent MAP kinases, p38
MAP kinase, and IKBkinases (296-298,300).
Recently, the PEA3 family of transcription
factors as well as NFKBp65 have been shown
to increase COX-2 mRNA levels (301). PPAR y
has been shown to suppress LPS induction of
COX-2 in macrophages (302). In addition, p53
has been shown to be a transcriptional inhib-
itor, explaining the expression of COX-2 in tu-
mor cells that in many cases lose p53 (303).
Associated with COX-2 expression in tumors is
the report that k Ras, a protein associated with
tumors, increases the stability of COX-2 mRNA
(304). The anti-inflammatory activities of salic-
ylate and corticosteroids are at least partly at-
tributable to suppression of COX-2 expression
by suppression of transcription and mRNA de-
stabilization, respectively (288).
In several cellular systems, higher levels of
prostaglandin production have been associ-
ated with COX-2 expression. In some cells (e.g.
WISH) expression of both enzymes yields
prostaglandin production from only COX-2, as
shown by specific inhibitors (Hulkower and
Bell, unpublished observations, 1996; 305). In
addition COX-1 appears to prefer exogenous
arachidonic acid, whereas with endogenous
substrate COX-2 metabolism is predominant
(298). These phenomena have three possible
explanations: (1) specific linkage of each en-
zyme with a specific phospholipase, (2)linkage
of COX-2 to an inducible PGE, synthase, and
(3) regulation by peroxide tone.
A number of enzymes have been linked to
arachidonic acid release and subsequent pros-
taglandin formation. Types 11, V, and IV
PLA,s have been linked to prostaglandin re-
8 Biochemistry and Structural Biology of Cyclooxygenases
lease as well as phospholipase Cldigyceride
iipase and phospholipase D (306). The enzyme
responsible for release of substrate may be
stimulus and cell type dependent. In the mast
eell early phases of AA release are dependent on
a phospholipase different from that in the sec-
ondary phase (307). A number of workers have
euggested specific phospholipase activation cou-
pled to either COX-2 or COX-1 (308,309).
Very recently, two laboratories have made
progress in identifjing the enzymes responsi-
ble for PGE, production from PGH,. Two en-
zymes have been identified. The first is a cyto-
solic, constitutively expressed enzyme and
appears linked to COX-1 (308,310). The other
enzyme is microsomal in location, is inducible,
and appears linked with COX-2 PGE, produc-
tion (310-313). The importance of the induc-
ible PGE, synthase in control of the relative
contribution of COX-1 versus COX-2 PGE,
production remains to be thoroughly under-
hood but has significant potential, perhaps
even as a new drug target.
Finally, a third molecular mechanism for
the difference between the two COX enzymes'
turnover and activation rate has been pro- - prostacyclin production is less clear with both
posed. Elegant kinetic studies show clearly
that the two enzymes differ in the peroxide
reaction kinetics of stabilization of the active
cyclooxygenase species. The interpretation of
these studies is that a lower level of peroxide is
required for COX-2 turnover than that for
COX-1. Practically, this means that under
some cellular conditions COX-1 is silent,
whereas COX-2 produces prostaglandins in
significant amounts (291).
A final aspect of cyclooxygenase biosynthe-
3is of prostaglandins is the cellular location of
the enzymes. A number of investigators (280,
290) have described a preference for COX-2 to
localize to nuclear membranes compared to
COX-1, although this is cell and stimulus de-
pendent. Recent publications have also de-
mibed COX-2 location in lipid bodies and in
caveoli (314).
8.6 Actions of Cyclooxygenase-Derived
Eicosanoids
8.6.1 Homeostatic Effects of Prostaglandins.
k large number of organ systems are influ-
rnced by prostaglandins. These include car-
233
diovascular, gastric, kidney, and reproductive
organ systems. Recent research, takingadvan-
tage of the availability of selective COX-2 in-
hibitors and reagents differentiating the ex-
pression of the two enzymes, has begun to
detail the relative contribution of the two en-
zymes in these organs and their function.
The clearest function is in the gastrointes-
tinal (GI) tract, where prostaglandins clearly
play a protective role, as evidenced by the su-
perior safety of COX-!&selective agents and
the protective effects of misoprostal on the GI
lining. The primary drawback of traditional
nonselective agents is in the gastric system,
where reduction of protective prostaglandins
causes ulceration, bleeding, and occasionally
death.
Cardiovascular effects of prostaglandins
are more complex. The coagulation system is
clearly modulated by platelet-derived throm-
boxanes, which have procoagulation effects
and the anticoagulative effects of endothelial
cell-derived prostacyclin. Thromboxanes are
clearly COX-1 derived because platelets do not
express COX-2. The source of endothelial cell
enzymes expressed and mixed opinions on the
relative contribution of the two enzymes. Re-
cent results with Vioxx, a selective COX-2 in-
hibitor, imply a modest effect on myocardial
infarction rate in patients taking the com-
pound but do not define whether this is caused
by a change in ratio simply by a lack of platelet
effect or that inhibition of endothelial cell
prostacyclin also skews the pro- and anticoag-
ulant ratio.
Prostaglandins regulate renin-angiotensin
secretion and thus glomerular filtration rate
and sodium homeostasis. These effects appear
to be COX-2 driven (315). The kidney is a rare
organ, one that expresses COX-2 under non-
pathological situations. Expression in the loop
of Henle apparently drives prostaglandin for-
mation in the kidney and the subsequent
physiological responses. Thus a selective
agent would likely have similar negative ef-
fects on kidney function as those of the nonse-
lective NSAIDs. This is apparently the case
with both Vioxx and Celebrex (315).
Prostaglandins play important roles in
each step of mammalian reproduction. These
prostaglandins appear to be COX-2 derived, as
 COX-2 Inhibitors and Leukotriene Modulators

delineated by COX-2 null mice (316). Thus the
reproductive effects associated with NSAIDs
would be expected to occur with COX-2-selec-
tive agents as well.
8.6.2 Pathophysiological Effects of Prosta-
glandins. Nonselective COX inhibitors, the
NSAIDs, have long been used as anti-inflam-
matory, antipyretic, and analgesic agents.
Studies in animals and in humans show
clearly that selective COX-2 inhibitors such as
rofecoxib and celecoxib are as effective in
these areas as earlier nonselective agents.
These results strongly indicate that the major-
ity of the prostaglandins causing inflamma-
tion, pain, and fever are COX-2 derived.
Older nonselective agents and more re-
cently new selective COX-2 inhibitors have
been shown to have anticancer effects in ani-
mal models and some human experience
(317). These studies imply effects of prosta-
glandins on several stages of cancer progres-
sion. Early studies have shown prostaglandins
to be important as modulators of cell prolifer-
ation (318) and, not unexpectedly, COX-2 in-
hibitors have shown pro-apoptotic effects
(319). More recently, expression of COX-2 has
been shown to be cell cycle dependent in hu-
man fibroblasts (320) and associated with G1
delay in intestinal epithelial cells (321). Even
earlier in cancer cell progression than tumor
cell growth, COX-2 inhibitors block carcino-
genesis in animal models (322). Finally, COX-
2-selective agents block later stages of tumor
growth such as angiogenesis (323). Most sig-
nificantly, it was shown recently that overex-
pression of COX-2 in mice was sufficient to
cause tumorigenicity (324).
9 SELECTIVE COX-2 INHIBITORS
nism for cyclooxygenase inhibition. Several
reviews have addressed various aspects of the
COX-2 story (428), a very recent review (386)
and two minireviews or summaries (398,399)
are cited within the following discussion on
this section.
9.2 Structure-Activity Relationships
9.2.1 CSulfonylphenyl COX-2 Class. The
most investigated area of selective COX-2 in-
hibitors is the 4-sulfonylphenyl super genus or
family, to which both Celebrex (celecoxib)and
Vioxx (rofecoxib) belong (325, 326). Often re-
ferred to as the "tricyclic," "diaryl," or "cis-
stilbene" class, the lead structures for this
general class were a series of known diaryl anti-
inflammatory agents. The most referenced lead
structure appears to be DuP 697 (49) (327).
(49) DuP 697
The development of this compound was in
part driven by the observation of less gastro-
intestinal irritation in animal models. A struc-
tural representative of this early class of anti-
inflammatory agents, predating DuP 697, is
illustrated by flumizole (50) (328). After the
discovery of the inducible COX isoform, ana-
logs having a methanesulfonylphenyl moiety

9.1 Agents Inhibiting COX-2

The hypothesis that a selective COX-2 inhibi-
tor would be safer and therefore provide an
attractive alternative to NSAIDs has been val-
idated by the clinical successes of Celebrex and
Vioxx. Both of these agents are from the larg-
est or most studied class of COX-2 inhibitors,
although other classes have been identified
and each has characteristic kinetic and ther-
modynamic parameters describing a mecha- (50) Flumizole
9 Selective COX-2 Inhibitors
as one of the aryl rings were quickly segre-
gated as a subclass having high COX-2 selec-
tivities. The 4-methylsulfone or 4-sulfon-
amide-substituted phenyl ring is the hallmark
ofthisclass of selective COX-2 inhibitors. Sub-
sequent crystallographicstudies of the protein
with inhibitors have provided the rationale for
this structural requirement (285, 329-331).
Initial studies showing that mouse and human
enzyme proteins were quite similar were fol-
lowed by an X-ray structure of SC588 (51c)
cocrystalized with mouse COX-2 (329). These
studies revealed an extra side pocket created
by an isole~cine/valine~~~ switch and that this
new "nook" apparently is engaged in binding
of the methylsulfone moiety and provides an
opportunity for COX-2 selectivity. This site is
the most prominent difference in the COX-1
vs. COX-2 active-site comparisons. This differ-
ence provides a key design element of in-
creased steric bulk to augment COX-2 selec-
tivity. SAR studies reported for celecoxib
(Ma) and rofecoxib (52) illustrate many gen-
eral characteristics of this class.
(51) Searle Series
(51a) R1 = NH3, Rz = CH3 Celecoxib
(51b) R1 = CH3, Rz = C1 SC-263
(51c) R1 = NH3, R z = Br SC-588
(51d) R1 = NH3, Rz = F SC-58451
(51e) R1 = CH3, Rz = F SC-58125
(52) Rofecoxib (MK 966)
The common pharmacophore shared by
these two structures and this class consists of
two vicinal substituents being attached to ad-
jacent sp2 atoms of a five-membered heterocy-
cle or carbocycle. One of the two must be a
4-sulfonylphenyl moiety and the other is often
a second substituted phenyl ring. This concept
has been applied very successfully to many
heterocycles and confirmed the generality of
this scaffold or template model for this class.
Analogs using the 1,5-diarylpyrazole (51, 57)
of celecoxib (51a)as a scaffold as well as re-
lated 3,4-diarylpyrazoles (332), 1,Sdiarylpyr-
roles (63, 64, Table 5.1), l,2-diarylimidazoles
(59,Table 5.1) (334),4,5-diarylimidazoles (58)
(335),4,5-diarylthiazoles(60) (336, 337), 4,5-
diaryloxazoles (332, 338), and 3, 4-diarylisox-
azoles (valdecoxib, SC-65872, Bextra, 53) have
(53) Valdecoxib SC-65872
been thoroughly studied (339,340). In addition
to the 2(5H)-furanone sdfold of rofecoxib (52),
related cyclopentenone (341), 3(2H) furanone
(342), and 1,5-pyrrolin-2-ones (343) have also
been reported as closely related series based on
carbonyl-containing heterocyclic templates or
scaffolds. Other five-membered heterocycles
have been developed, including 3,4-diarylthiazo-
lin-2-one (344),4,5-diaryloxazolones(345,3461,
diarylthiadiazole (3471,and diaryltriazole (348).
Other core scaffolds or templates have also been
developed from modified five-membered hetero-
cycles that have been fused to a second cyclic
ring (349351). Many examples of these het-
eroaryl-bicyclic templates have been reported,
including FR-228352 (54, reported to be in
phase I testing (340). Six-membered heterocy-
clic d o l d s have also been shown to be effec-
tive and include pyridine (352) [etoricoxib, (55)
(353)],pyridazinone (354), and pyranone (355).
236 COX-2 Inhibitors and Leukotriene Modulators

Table 5.1 COX Activity/Selectivity of Selected Inhibitors

Heterocyclic
Template COX-1 (/Lao COX-2 (/Lao R Reference
(57a) 25.5 0.41 NH2 325
(5%) 0.08 0.01 NH2 325
(58a,b) >lo0 0.19 CH3 335
(59a) >loo 5.85 CH3 334
(59b) 36 0.1 CH3 335
(60a) >loo 0.023 (3% 337
(60b) 1.07 0.006 CH3 337
(61a) (DuP697) 0.6 0.005 cH3 364
(61b) 1.1 0.02 CH3 364
(62a) >lo0 0.25 CH3 364
(62b) >50 4.3 CH3 364
(63a) >loo 0.06 CH3 333
(63b) >I00 >lo0 '333 333
(64a) >loo 0.51 CH3 333
(64b) >100 10.2 cH3 333

Five- and six-membered carbocycles have
also been incorporated as efficient scaffolds.
Examples using phenyl (known as terphenyls)
(356,357),cyclopentene (356,358-362), cyclo-
pentadiene (359), cyclobutenone (3631, and
naphthyl (356) cores or scaffolds have been
described. The cyclopentene series was opti-
mized to a high degree, producing many very
potent and selective COX-2 inhibitors with
good in vivo activity (e.g., 56, SC-57666).
Other structural elements on the scaffold
may be relevant in relation to the regiochem-
istry of the two key adjacent substituents on
the template. Minor changes in positioning
the two aryl rings can result in dramatic dif-
ferences in selectivity between two very simi-
lar isomers on the same template. Table 5.1
shows data from several analogs where the rel-
ative positions of the two key substituents
have been interchanged on a given template.
In the 3-trifluoromethylpyrazole template, or
celecoxib series, the 4-sulfonylpheny moiety
must be attached in a 1,3 relationship relative
to the 3-trifluoromethyl substituent of the
pyrazole, as in both ( 5 7 4 and (57b)(Fig. 5.4).
Both of these isomers are potent COX-2 inhib-
9 Selective COX-2 Inhibitors

(58);these isomers have the same 1,3 relation-

ship of the 4-methanesulfonylphenyl and the
3- CF, substituents. Scaffold symmetry elimi-
nates further analysis, given that the isomers
with respect to the imidazole NH cannot be
resolved. The 1,2-4-trifuoromethylimidazole
(59a, 59b; Fig. 5.4) shows a somewhat differ-
ent result in terms of selectivity relative to the
1,5-pyrazoleof the celecoxib series (37a, 3%).
The isomer (59b), which is more potent at
both isoforms, turns out to be the more selec-
tive because of an increase in affinity at
(55) etoricoxib (MK-0663)
COX-2. When applied to the 4,5-thiazole (60a,
60b) scaffold, the 6sulfonylphenyl moiety ad-
itors. One isomer (57a) has superior selectiv- jacent to the nitrogen of the heterocycle,
ity. The isomer with the 4-sulfonylphenyl ring (60a), is much more selective than the other
in the 5-position and F-phenyl in the l-posi- (60b) (Fig. 5.4) isomer by virtue of a much
tion (57b)had higher affinity for COX-2 but weaker COX-1 activity and little change in the
was less selective because of a dramatic simul- COX-2 potency. Reported SAR for the thio-
taneous increase in COX-1 potency. phene series reveals a much smaller difference
e 4,5-diaryl-3-trifluoromethylimdazole in potency and overall COX-2 selectivity be-
Id produces a selective COX-2 inhibitor tween isomers (61a and 61b).This template

A~ Arl&*
/
H$A
Ar1
N*

0 /
A12 3 Ar2 3 A r2

=reF
65 a, b 66 a, b 67 a,b

(a) Arl = - $0s02R Ar2=

(b) Arl = = $ O F \ /
Ar2= - $ e S 0 2 R

Figure 5.4. Carbonyl containing heterocyclic templates.

 COX-2 Inhibitors and Leukotriene Modulators

has the sulfur heteroatom as well as the

2-bromo substituent as structures introduc-
ing template asymmetry, but shows little pref-
erence with respect to the relative 4,5 position
of the 4-methylsulfonephenyl moiety. For
comparison, in the thiophene template with-
out the 2-bromo substituent (62a, 62b) an ap-
proximately 2 log shift to the right in COX-1
potency is observed but only about a 10-fold
shift in COX-2 potency for the "a" isomer
(62a), which has the most "useful" selectivity
of the 4-thiophene compounds shown. Of the
two possible isomers of the 5-methyl pyrrole
scaffold, the "a" isomer (63a) has good COX-2
selectivity, with the "b" isomer (63b) being
one of the few analogs completely inactive at
COX-2. The unsubstituted pyrrole (64a, 64b)
exhibits a similar trend, with the a isomer
having superior activity at COX-2 and there-
fore being a selective agent, although the rel-
ative increases are dramatically different from
what was observed in the methyl-substituted
template (63).
A related phenomenon is the similar re-
quirement of a 1,3 relationship of the Csulfo-
nylphenyl ring and a structural feature of the
scaffold. In the rofecoxib series, the relative two key aromatic substituents on the scaffold,
position of carbonyl of the furanone is critical. is currently not well developed. Although X-
The analog (65a) (Fig. 5.4), where the rel- ray structures and modeling techniques have
ative position of the Csulfonylphenyl ring and provided valuable gross features of the en-
the carbonyl are 1,2, is inactive at both iso- zyme, to date a large number of X-ray struc-
forms; the reverse orientation, (65b), is of tures with bound ligands have not been avail-
course the highly selective rofecoxib template. able. The scaffold model does seem valid,
Similar to the rofecoxib series, the 4,Bdisubsti- based on the wide range of heterocycles that
tuted pyridazinones (365) show a required 1,3 have been used and, with a few exceptions (see
relationship of the sulfonylphenyl moiety and 65a and the other non-l,3-carbonyl analogs
the template carbonyl. Of the possible four iso- 66a and 67b), most produce potent COX-2 in-
mers, only two analogs (66b and 67a) (Fig. 5.4) hibitors with less potent COX-1 activity.
are potent selective COX-2 inhibitors. One of the only successful modifications for
Broader variations on the scaffold model the methylsulfone moiety has been the unsub-
have been reported in the patent literature. A stituted sulfonamide. Both sulfonamide and
prodrug of the rofecoxib furanone template sulfone analogs can also be quite potent
has been reported (366). The cis-stilbene-like against COX-1, however. The extra isoleucinel
structure (68) can undergo metabolic oxida- dine5', space is limited, even in COX-2, and
tion to produce the selective 2,3-diary fura- the SAR for this substituent is very restrictive.
none. A variation on the 1,2 vicinal substitu- For example, the N-methyl sulfonamide in the
tion is the reported analog (69), with geminal celecoxib series was inactive (325). Reversal of
positioning of the two aryl rings off the exocy- the N and SO, atoms gave an inactive com-
clic olefin of the butyrolactone template (367, pound. The introduction of other strong elec-
368). tron-drawing group replacements for sulfonyl
The rationale for these related phenomena, like nitro or COCF, lead to inactive com-
involving the critical relative positions of the pounds at COX-2. The 4-nitro analog (70) did
9 Selective COX-2 Inhibitors
CF3
lave some COX-1 activity (IC,,, COX-1, 1.75
phfl (325). Only a few highly COX-1-selective
inhibitors have been reported, which are often
found as part of SAR analysis of sulfonyl re-
jlacements. Examples of very selective COX-1
analogs from the pyrazole (71) (325) andI thio-
phene (72) (364) series have been identified.
CF3
F stronger electron-donating groups, such as
A significant empirical trend has been iden-
ified by comparison of direct sulfonamide and
ulfone analogs. The sulfonamide analog is
pnerally more potent but this usually applies
o both isoforms. In many cases the COX-1
lotency is often disproportionately increased,
3ading to a decreased selectivity ratio (332,
359-361,364). The sulfonamide can have su-
perior bioavailability and physicochemical
properties that are manifested in greater in
vivo efficacy in the anti-inflammatory models.
Celecoxib has incorporated the sulfonamide as
a balance of selectivity and in vivo efficacy.
The JTE-522 compound (73)introduced a flu-
(73) JTE 522
orine ortho to the sulfonamide and reported
the compound with this modification regained
COX-2 selectivity more characteristic of the
methylsulfone analog (369, 370).
In contrast to the restrictive nature of al-
lowed modifications for the 4-sulfonylphenyl
substituent, a wide variety of structures can
be incorporated to replace the second phenyl
ring. 4-Halogen-substituted phenyl is the
most used reference substituent for this posi-
tion. The early Searle candidate, SC-263 (51b)
with a 4-C1 substituent was found to have an
unacceptably long half-life, which can also be
characteristic of this class. In celecoxib (51a),
introduction of the para-methyl group on this
ring, as a site for metabolic oxidation, reduced
the rat plasma half-life from 221 h for SC-263
(51b)to 3.5 h for celecoxib (51a)(325). Clini-
cal trials with DuP 697 also identified its ex-
tremely long half-life of 292 h as a develop-
ment issue (371). In general halogen and small
alkyl groups are well tolerated on this ring;
methoxy, are tolerated in the 4-position as
well. Other halogen effects on this ring are
noted in the rofecoxib derivative DFU (74)
[the name DFU appears to be an acronym re-
lated to the 5,5-dimethy-2(5H)-furanone scaf-
fold] (372, 373). Improved selectivity can in
part be attributed to the introduction of the
ortho fluorine atom into the non-sulfone-bear-
ing phenyl ring. Dihalogen substitution also
frequently gave compounds with a 1000-fold
in vitro selectivity (325). SAR studies indicate

(74) DFU
many heterocycles to be bioisosteric, with phe-
nyl for many templates (337, 352, 374). The
traditional, older diary1classification or termi-
nology to describe this class has been sup-
planted by demonstration that the non-4-sul-
fonylphenyl vicinal substituent can be a
number of lipophilic as well as polar nonphe-
nyl substituents. JTE-522 (73) was one of the
first widely referenced analogs with a cyclo-
hexyl ring in place of substituted phenyl(369).
DFP (75) (DFP is also a possible acronym
indicating propyloxy substitution on the di-
(75) D F P
methyl-furanone scaffold), a potent analog of
DFU, introduced methylpropoxy subtitution
for phenyl and maintained COX-2 selectivity
and potency (375). In addition to alkoxy,
thioalkoxy and aminopyridyl substitution ad-
jacent to the sulfonylphenyl ring using a pyri-
dine ring central scaffold were reported to pro-
duce potent COX-2 inhibitors (376). The
presence of these polar side chains was pro-
posed to improve oral bioavailability through
improved absorption. Arylpyridazinones have
also been shown to tolerate 3-methylbutoxy
and 2-methylpropoxy substituents adjacent to
COX-2 Inhibitors and Leukotriene Modulators
the phenylsulfonyl substituent (354). Even
more polar substituents, such as alkoxyhy-
droxy substitution, have been reported to
maintain good COX-2 selectivity in the pyri-
dine scaffold series (376).
The Abbott compound ABT-963 (76)has a
3-hydroxy-3-methylbutyloxysubstituent adja-
cent to the 4-methanesulfonylphenyl moiety
and shows improved selectivity as well as ex-
hibiting superior solubility, protein binding
and pharmacokinetics (365). Additional tern-
plate or scaffold substituents, beyond the well-
studied 4-methanesufonylphenyl and its 1,2
vicinal partner, are limited and are more spe-
cific to the template or scaffold. In the cele-
coxib series the 3- CF, pyrazole substituent
appears optimized, although a wide variety of
substituents were tolerated (325). Several
substituents, including hydroxymethyl, cya-
nomethyl, benzyloxymethyl, Cmethoxyphe-
nyl, and 5-chloro-2-thienyl in the 3-position,
were shown to maintain potency and selectiv-
ity comparable to those of the CF, analog. The
hydroxymethyl-substituted analog was active,
similar to a reported hydroxymethyl metabo-
lite of valdecoxib that was isolated and re-
ported to have a potent selective COX-2 activ-
ity as well as oral anti-inflammatory activity
(339). Some minimal size or specific electronic
nature of the 3-pyrazole substituent appears
to be important, given that the 3-methyl ana-
log of celecoxib displays very weak COX-2 ac-
tivity and the 3-unsubstituted analog is corn-
pletely inactive (>I00 f l at COX-2). This
example illustrates one of the weaknesses
of this heteroaryl template (347) general
strategy.
A small change in the template can render
it a poor scaffold. The 1,3 relationship of the
9 Selective COX-2 Inhibitors

3-trifluoromethyl substituent and the 4-meth-

anesulfonylpheny substituent on the pyrazole
g is an example of an optimized template.
an attempt to survey a large number of five-
membered templates to find a replacement for
the thiophene template, the unsubstituted
4,5-diaryloxazole was discarded for lack of po-
tency (347).The 4,5-diaryl-2-methyloxazole is
a template that produces selective analogs and
is found in JTE-522 and, like the 1,5-diaryl-
pyazole scaffold of celecoxib, requires an addi-
tional small substituent for maximun activity.
A series of 1-arylpyrrolin-2-ones (343) em-
ploys an additional aryl substituent on the
scaffold similar to the pyridazinones (354).
This class of COX-2 inhibitors is generally
pophilic and lacks water solubility; the for-
mulation of a parental or i.v. administrable
COX-2 inhibitor has been addressed by Phar-
macia in development of a water-soluble pro-
drug of valdecoxib (531, known as parecoxib

AYo
sodium (77) (340, 377).

this compound (79) is reported to be 33-fold

selective for COX-2.

9.2.2 N-Arylsulfonamides. Lead structures

for this class of selective COX-2 agents were
Na 0 also compounds developed as anti-inflamma-
tory agents before the discovery of the COX-2
H 3 C0~ N \ 0
! - isoform. Nimesulide (R-805,80)was identified

(77) Parecoxib Sodium

This compound is derived from acylation of

e sulfonamide moiety of valdecoxib, followed
treatment with sodium hydroxide. The acidic
acylsulfonamide forms a water-soluble sodium
salt that is stable for administration but cleanly
delivers valedecoxib once in plasma.
Related methanesulfonylheteroaryl inhibi-
tors (78 and 79) from Chugai are reported in
the patent literature (378). These compounds
ay be interacting in a manner similar to that
of the methanesulfonylphenyl inhibitors, al-
though they do not fit the general model be-
cause there is no substituent adjacent to the
sulfonyl group on the aromatic scaffold. The
direct attachment of the methanesulfonyl
group to a nonphenyl ring appears to be novel.
Both cores reported are indole derived and
(80) nimesulide (R-805)
as an NSAID anti-inflammatory drug with
weak prostaglandin synthetase activity (COX-1)
but potent in vivo in the carrageenan-induced
edema model (379). An analog of nimesulide,
from Taisho, NS-398 (81) was shown to be a
selective COX-2 inhibitor (380). This class has
a characteristic acidic proton by virtue of the
N-arylsulfonamide or methanesulfonanilide.
A related analog, FK-3311(83),incorporated a

methyl ketone as an alternative to the nitro
electron-withdrawing group and introduced
2,4-difluoro substitution into the phenoxy
ether ring (381). This series was expanded to
show that thiophenoxy ethers were as potent
as the oxygen analogs. Flosulide (GP-28238,
84) incorporates a difluorophenoxy substitu-
(84) Flosulide (CGP 28238)
ent ortho to the sulfonanalide and introduces a
modified electron-withdrawing keto group, as
part of a conformational restricted fused cy-
clopentanone (382).
Flosulide was shown also to be a selective
COX-2 inhibitor, less potent than NS 398 but
with better pharmacokinetic properties (383).
Flosulide was used as a lead for development
of L-745,337 (85), which is the direct thio-
COX-2 Inhibitors and Leukotriene Modulators
ether congener of Flosulide. L-745,337 is more
active than Flosulide in the rat paw edema in
vivo assay (383,384). The SAR for this series is
reported to be very limited with cyclohexyl,
thiazole, and to a lesser extent pyridyl being
some of the very few substituents that can re-
place the phenoxy ring (385). This restrictive
SAR may also explain the lack of further re-
ported development of this series.
9.3 Selective NSAlDs and NSAlDs Modified
for Improved COX-2 Selectivity
Given the homology between the two cy-
clooxygenases, the reexamination of known
anti-inflammatory agents, with particular in-
terest in reported PGHS inhibitors, is an area
of research regenerated by the identification
of the inducible COX-2 isoform. Some of the
most selective NSAIDs, such as diclofenac,
have COX-1ICOX-2 selectivities in about the
threefold range (386). However, tomexiprole
(86) was reported to have 30-fold COX-2 selec-
(86) tornoxiprole
tivity (387). Surprisingly, etodolac (87) has
been reported to be as selective as the rofe-
coxib derivative DFU (COX-l/COX-2, 1000x)
9 Selective COX-2 Inhibitors
(87) etodolac
re-
Other NSAIDs with weak COX-2 selectiv-
ity. like meloxicam. have been modified, with
considerable structural reorganization,
yield structures like (88)with enhanced se
The model of tlie COX-2 protein as having a
larger binding site has been used for rational
design of superior COX-2 selectivity into exist-
ig NSAIDs. In addition to modeling studies,
supporting evidence has been the observation
after acetylation by aspirin, COX-2 ,still
oduces large amounts of 15-HETE after ad-
dition of AA (391). This activity was not ob-
served with COX-1, which is inactivated by
aspirin acetylation. The rationale that follows
.
im~liesthat the COX-2 Docket of o~ening.
A ., is
ge enough, even after acylation of key reesi-
not
have steric access to the smaller COX-1 pocket
aRer acetylation. Two types of modifications
of indomethacin (89) have been shown to in-
crease COX-2 selectivity. Modifications to the
N-benzoyl moiety, including trichloro substi-
(89) indomethacin
tution or conversion to 4-bromobenzyl, have
improved selectivity (391). A second, more
general modification has been the homologa-
tion or extension of the carboxyl functionality
in combination with alkyl branching, produc-
ing selective inhibitors like L-761,000 (90)
(392).
The alkyl branching on the carbon frame-
work extending the acid primarily blocks sites
of oxidative metabolism. Arnides and esters
formed using bulky or substituted alcohols
and amines have also produced indomethacin
derivatives like (91), with improved selectivity
through structural extension of the carboxy-
late domain (393). Incorporation of the car-
boxylate of indomethacin into the latent car-
bony1 of a thiazole ring (92) as a way to extend
the structural framework and take advantage
of the larger binding cavity of COX-2 is a re-
lated approach (394).The carboxyl functional-
ity of zomepirac has been extended as a sulfon-
amide (931, with improved COX-2 selectivity

(386). Flurbiprofen can be converted into a
more COX-2-selective agent (94) by addition
of steric bulk in the form of two ethoxy groups
to the terminal phenyl ring (395). No clinical
data have been reported with modified
NSAIDs thus far.
Selective compounds have been designed
de novo; the most selective analogs reported
COX-2 lnhibitors and Leukotriene Modulators
were also extended bulky amides like (95) and
presumably correlate to a difference in bind-
ing pockets (396). Modification of COX-1 bind-
ing ligands for greater COX-2 selectivity has
been successfully achieved and has been sup-
ported by structural information about the en-
zyme.
9.4 Irreversible lnhibitors
Aspirin is known to covalently acetylate the
Ser530residue in COX-1 and is the only known
NSAID to irreversibly inactivate COX-1 by
this mechanism. Acetylation by aspirin of the
COX-2 enzyme did not lead to inactivation but
did modify the product formed to 15-HPETE
(391). The acetoxyheptynyl sulfide (96), has
9 Selective COX-2 Inhibitors
been developed as a selective COX-2 inhibitor,
which similar to aspirin covalently acetylates
Ser516in COX-2 (397).
Unlike aspirin, this compound is reported
to selectively acetylate the COX-2 serine resi-
due and selectively inactivate COX-2. The
bulky heptynyl side-chain was optimized for
COX-2 selectivity in this S A R study and the
selective inhibition reported must be related
to the differences in active site or binding
pocket size, similar to the strategy for creating
many other selective inhibitors from modifica-
tions of COX-1 inhibitors.
9.5 Biochemical Assays, Selectivities,
and Potencies
One of the challenges associated with evaluat-
ing COX-l/COX-2selectivity is trying to define
or identify accurate affinities or dissociation
constants for compound comparison. Inhibi-
tory concentration data against purified pro-
tein obtained from baculovirus or CHO cells
transfected with COX-1 or COX-2 have pro-
vided a majority of the information with re-
gard to relative potency of individual agents. A
complicating factor is that inhibitory activity
exhibits a characteristic time-dependent phe-
nomenon with many classes of COX-2 inhibi-
tors; only under the simplest kinetic condi-
tions, do K, values give good approximations
of equilibrium dissociation constants that are
needed to facilitate traditional S A R studies. In
addition, the stable cell lines expressing the
two enzymes must be stimulated with exoge-
nous AA, quite unlike the cellular situation
where AA is released in smaller concentra-
tions internally. Typical values reported for
highly selective compounds against broken
cell or transfected CHO cells are micromolar
COX-1 potencies and low nanomolar potencies
for COX-2 activity, giving approximately
1000-fold selectivity ratios.
Celecoxib has IC,, values of 40 nM and 15
for COX-2 and COX-1, respectively,
against the recombinant human enzyme assay
(325). Rofecoxib has IC,, values of 20 nM and
>15 for COX-2 and COX-1, respectively,
using protein from the CHO cell assay (326).
Some of the nonselective reference com-
pounds are quite potent in COX-2; IC,, values
reported for indomethacin range from 30 to
900 nM for COX-2 in the broken cell assays
(380, 326) and 10 to 50 nM for COX-2 in the
engineered CHO cell (383, 392). DuP 697 is a
potent COX-2 inhibitor in the CHO cell, with
an IC,, value of 2-10 nM (383, 363). It is also
COX-2 selective, having a weaker correspond-
ing COX-1 IC,, (59-100 nM). The most potent
COX-2 IC,, values are single-digit nanomolar
and many of the cyclopentene derivatives ap-
proach this maximal activity (360, 362). The
furanone derivatives appear to have a slightly
weaker potency against COX-2 but are also
much weaker against COX-1, resulting in sim-
ilar selectivity ratios.
Later reported data indicate most compa-
nies switching to human whole blood (HWBL)
assays for better indications of the effects of
protein binding and other pharmacokinetic
parameters considered relevant in correlating
in viuo potencies and selectivity. Most of the
reported compounds, including celecoxib and
rofecoxib, are much weaker in the HWBL as-
says, with potencies on the order of 0.5-1 @.
Merck reports an IC,, value for rofecoxib in
the human COX-2 HWBL assay of 0.5 @,and
in the corresponding COX-1 HWBL assay, an
IC,, value of 19 pM (38-fold selectivity) (326,
353). Comparison values for celecoxib are re-
ported to be an IC,, of COX-2 HWBL assay of
1.0 @ and the corresponding COX-1 HWBL
assay, an IC,, value of 6.3 @ (-sixfold selec-
tivity). In this same comparison the JTE-522
compound was found to have HWBL potencies nr
of approximately 33 and 100 @ for COX-2
and COX-1, respectively, for an approximate I:
'i
only threefold selectivity. More recent agents
such as etoricoxib (Arcoxia), valdecoxib (Bex-
tra), and ABT-963 have greater reported selec-
8
tivity in the human blood assays compared to @
that of rofecoxib and celecoxib.
One of the most important mechanistic
characteristics of COX-2 inhibition exhibited
by many COX-2-selective inhibitors is the ob-
served time-dependent inhibition of COX-2
but not of COX-1. A slow, noncovalent,
pseudo-irreversable conformational change in
the protein occurs after binding of the inhibi-
tor, but this is only observed in binding with
the COX-2 enzyme (353,398,399). As a result,
a much lower concentration of inhibitor is re-
quired for effective inactivation of the COX-2
enzyme after a longer incubation, even for a

compound with equal dissociation constants
at the two isoforms. This time-dependent in-
hibition of prostaglandin biosynthesis by cer-
tain NSAIDs was examined by Lands and his
analysis showed that 0.3 $ flurbiprofen (an
NSAID that shows this time-dependent phe-
nomenon associated with a second conforma-
tional change after binding) could over a short
time period compete with 50 $ibuprofen (a
freely reversible NSAID), even though they
both had similar dissociation constants (400).
In effect, this boosts the apparent selectivity
for COX-2 of any inhibitor that can induce this
COX-2-selective and time-dependent confor-
mational change. This appears to be an im-
mense advantage to this type of inhibitor and
would argue that a lower selectivity could be
tolerated, as long as the COX-1 activities were
relatively high. Additional detailed models on
the complex kinetics of COX-2 binding have
been proposed (401).
9.6 In Vivo Models and Corresponding
Efficacy
Several models of inflammation and analgesia
have been used to characterize the in vivo
pharmacology of COX-2-selective inhibitors.
In addition to efficacy in inflammation and
pain models, models of gastric irritation have
been used to define superior safety indices for
these agents. ,lCr excretion in the rat has
been used most often for a gastric safety
model, with selective COX-2 inhibitors show-
ing no leakage from possible intestinal lesions
at doses often starting around 100 m a g
(358). For acute inflammation the rat carra-
geenan paw edema model is most often used.
ED,, values for celecoxib and rofecoxib in this
model are 7.1 and 1.5 mg/kg, respectively (325,
326). Indomethacin (375, 326) and DuP 679
(327, 363) are very potent in this assay, with
ED,, values around 1 m a g . Rofecoxib and
related furanones seem slightly more potent
in this assay than the celecoxib pyrazole or
related templates, including the cyclopentene
series. Flosulide was reported to have one of
the lowest ED,, values reported for this assay,
with an ED,, value of less than 1 mg/kg (0.6
mg/kg) (383).
In models of chronic inflammation, for
which the rat adjuvant arthritis assay is fre-
quently used, these selective COX-2 inhibitors
COX-2 Inhibitors and Leukotriene Modulators
are very potent. ED,, values of 0.1 to 1 mg/kg
are achieved with analogs from many different
series; representative efficacies are as follows:
celecoxib, ED,, 0.37 m&g (325); rofecoxib,
ED,, 0.15 mg/kg (326); DuP 697, ED,, 0.18
mg/kg (379); and indomethacin, ED,, 0.11
m&g (325). COX-2 inhibitors have also
shown excellent analgesic activity. Hyperanal-
gesic paw models, either yeast-induced (Ran-
dall-Selitto) or carrageenan-induced (Har-
graves), are most commonly reported as a
measure of analgesic activity. Another model
that is often reported is the rat air pouch in-
flammation model. This model is reported to
measure anti-inflammatory events at the site
of inflammation. The cyclooxygenase inhibi-
tors are also very potent in this model, often
giving 90% and greater inhibition at 2 mgkg
doses (R. Harris and R. Bell, personal commu-
nication, 1996).
9.7 Conclusion
Since the discovery of the COX-2 isoenzyme, a
significant number of chemical agents have
been synthesized with the goal of finding a
selective inhibitor. As just described, defining
what is meant by "selective COX-2 inhibitor"
in vivo has been difficult. This is not surpris-
ing, given the similarities of the active sites for
COX-1 and COX-2, complexities of the enzyme
reaction, the possibility for metabolism of
compounds, and so forth. As described in
greater detail in section 11, the search, al-
though difficult, has been successful. Clinical
examination of Celebrex and Vioxx have
shown a clear safety advantage for the com-
pounds, with no loss of efficacy.
10 AGENTS INHIBITING COX-2
AND 5-LIPOXYGENASE
Agents that can inhibit more than one inflam-
matory mediator may be advantageous. Com-
pounds that inhibit both COX-2 and 5-LO may
have an efficacy advantage over current
agents in the treatment of rheumatoid arthri-
tis. Parke-Davis has reported CI-1004, dar-
bufelone (98), and PD 138387 (97) as dual
5-LO/COX-2 inhibitors (402). Darbufelone
was reported to be in clinical trials and has
recently been described in detail (402). PD-
10 Agents Inhibiting COX-2 and 5-Lipoxygenase
(97) R = 0CH3, PD 138387
(98) R = H,(CI-1004)
138387 was developed by SAR studies and ex-
hibited superior selectivity to the earlier com-
PD-138387 (97) was reported to have an
0 nM against COX-2 and >10
th a modest oral ED,, value
15.7 mglkg. Analgesic activity was mea-
acid writhing in mice and
(Harris and Bell, unpub-
1997). Several structur-
related compounds have been reported
lfonamide into the het-
rocyclic moiety, for example, (99) (S-2474),
(99) 5-2474
ch has COX-2 selectivity of 2500-fold over
x-1 (cox-2, IC5, = 0.11 fl; cox-1,IC5,
el, an ED,, value of 0.76 mg/kg. Another
a series of phenoxy-substituted sulfon-
amido thiophene derivatives (100) (404). The
phenoxythiophene template has been used as
template for a series of N-hydroxyurea-based
5-LO inhibitors (131); the inclusion of the sul-
fonamide moiety results in compounds with
dual inhibition at 5-LO and COX-2. In uitro
activities of a preferred compound (100) were
reported as IC,, values of 1.97 for COX-2
and 0.73 pit4 for 5-LO. Other compounds have
been specifically designed by combining phar-
macophores from both COX-2 inhibitors and
5-LO inhibitors.
The patent report of a combined inhibitor
(101) (405) reveals the familiar oxazole (369,
340) COX-2 inhibitor attached to the Zeneca
methoxyphenylpyran, which was a very po-
tent binding element for a series of 5-LO in-
hibitors (155). In a similar manner, the ox-
azole COX-2 inhibitor has been linked to a
hydroxamic acid, which is also known to be a
potent iron ligand and primary pharmaco-
phoric moiety for many potent 5-LO inhibi-
tors, to give dual inhibitors like analog (102)
(406).The pyran-containing compounds (101)
exhibited potent and selective COX-2 inhibi-
tion (COX-2, IC,, < 0.1 a ; COX-1, IC,,, >
100 pik0 and 5-LO inhibition (IC,, = 0.02
f l .This compound exhibited oral in uivo ac-
tivity of 15% inhibition at 10 mg/kg in the rat
CPE model.

Thus far no clinical results with dual 5-LO/
COX-2 inhibitors have been reported.
11 CLINICAL EFFICACY AND SAFETY
OF SELECTIVE COX-2 INHIBITORS
Celebrex (celecoxib) and Vioxx (rofecoxib)
have progressed to registration and have es-
tablished the utility of this class of drugs in
treating the pain associated with surgery, os-
teoarthritis, rheumatoid arthritis, and muscle
pain.
Celebrex was the first COX-2 inhibitor to
be approved by the FDA (December 31,1998)
and Vioxx was approved somewhat later (May
22, 1999). More recently, Bextra (valdecoxib)
was approved (November 16, 2001) (Table
5.2).
The clinical trials, which were conducted
by SearlePharmacia to define the activity of
Celebrex, were extensive and involved nearly
16,000 subjects. Merck also examined a large
number of patients to define the activity of
Vioxx.
The first study by Hubbard et al. (407)
showed that Celebrex was effective in third
molar extraction, with significant difference
Table 5.2 Approved Eicosanoid Drugs
USAN Trade Name
Leukotriene
Zafirlukast Accolate
Zileuton ZYflo
Montelukast Singulair
COX-2
Celecoxib Celebrex
Rofecoxib Vioxx
Valdecoxib Bextra
Etoricoxib Arcoxia
V D A approval.
COX-2 Inhibitors and Leukotriene Modulators
from placebo but somewhat less effective than
ibuprofen. In another study, Celebrex at ei-
ther 100 or 400 mg was compared to aspirin,
and in this study all treatments were equally
efficacious and were significantly different
from placebo.
Malmstrom et al. (408) compared Celebrex,
Vioxx, and ibuprofen in an acute osteoarthri-
tis study and the treatments were rank-or-
dered as placebo, Celebrex, ibuprofen, and
Vioxx.
In a study examining the potential activity
of Celebrex in orthopedic surgery it was found
that, after a single dose, Celebrex was equal to
hydrocodone/paracetamol, but with multiple
doses, Celebrex was superior (409).
11.1 Osteoarthritis
The efficacv" of the COX-2 inhibitors in osteo-
arthritis was expected, given that classical
NSAIDs were effective in treating the pain as-
sociated with this degenerative disease. In a
study examining the effects of Celebrex in os-
teoarthritis of the hip, Geis et al. (410) showed
that 50, 100, and 200 mg doses were effective
in treating the pain in hip osteoarthritis and
the two higher doses were about equal to a 500
mg dose of naproxen. In a larger study exam-
ining the effects of Celebrex in osteoarthritis
of the knee (again where 50,100, and 200 mg
doses of Celebrex were compared to a 500 mg
dose of naproxen), significant improvements
were seen in the standardized scores in all
treatment groups; and at both the 100 and 200
mg doses the scores for Celebrex were better
than those seen with naproxen (411, 412). In
similar studies Merck also established the ef-
ficacy of Vioxx. Vioxx at doses of 12.5 and 25
Mechanism Date Approveda
CYSLT antagonist Sept. 1996
5-LO inhibitor Dec. 1996
CYSLT antagonist Feb. 1998
COX-2 selective Dec. 1998
COX-2 selective May 1999
COX-2 selective Nov. 2001
COX-2 selective Submitted 2002
I1 Clinical Efficacy and Safety of Selective COX-2 Inhibitors
mg resulted in improvement in both hip and
cnee osteoarthritis in studies that lasted from
5 to 86 weeks of duration (413,414).
Vioxx (50 mg) provided pain relief equal to
that of either 550 mg naproxen or 400 mg ibu-
profen (415, 416). In a multiple-dose study,
Vioxx produced significant pain relief after or-
thopedic surgery (416).
The clinical results with both Celebrex and
Vioxx suggest that inhibition of COX-2 is an
effective treatment for the pain that is associ-
ated with surgery, osteoarthritis, and rheuma-
toid arthritis.
Although most of the evidence suggests
that COX-2 is expressed only when induced by
cytokines or growth factors, there are poten-
tial roles this enzyme may play in normal
physiology such as in uterine contraction, re-
nal medulla, and in both brain and gut mu-
cosa. Extensive clinical trials with both Cele-
brex and Vioxx were conducted to determine
the safety of these compounds. Celebrex was
examined in 3- and 6-month trials and it was
found that the incidence of ulcers was the
same as that of placebo and significantly less
than that seen for either naproxen or diclofe-
nac (410). Vioxx was examined in 1516 pa-
tients using endoscopy, in either the 25 or the
50 mg dose, and was compared to ibuprofen
2400 mg. A significantly lower percentage of
ulcers were seen in the Vioxx-treated patients
than seen in the ibuprofen-treated individuals
(417). In another study, in which Vioxx was
given at 250 mg (10-20 times the clinical dose)
for 7 days, it was well tolerated, with gastric
injuries no worse than those of the placebo,
and was less than the injuries seen with either
2400 mg of ibuprofen or 2500 mg of aspirin
(418,411).
COX-2 is expressed in the renal medulla
and therefore there was concern that specific
COX-2 inhibitors could cause untoward renal
effects. However, renal effects of the com-
pounds have been reported to be mainly re-
lated to fluid retention in salt-depleted pa-
tients (420).
11.2 COX-2 and Cancer
Various studies have demonstrated a protec-
tive role of NSAIDs in the prevention of colon
cancer (421). However, the potential for gas-
tric damage intrinsic to conventional NSAIDs
precluded their use as preventive therapy.
Based on these observations, the scientists at
Searle/Pharmacia examined the potential role
of COX-2-selective inhibitors in first prevent-
ing polyps in mice and then in humans (422,
423). Data from the animal studies showed
that in rats treated with celecoxib and then
given s.c. injections of azoxymethane to induce
colon cancers, there was a 93% inhibition of
cancer incidence and a 97% reduction in tu-
mor number. Overall, there was a 87% reduc-
tion in the tumor burden (423). In the MIN
mouse model of familial adenomatous polypo-
sis (FAP),Jacoby et al. (424) showed that cele-
coxib inhibited the tumor number by 71% and
the tumor size by 83%. In chemically induced
bladder cancer in mice and rats, celecoxib was
effective at reducing the incidence of lesions in
both mice and rats.
Howe et al. (425) reviewed the potential of
using COX-2 inhibitors for the treatment of
breast cancer and suggested that there is good
rationale to examine COX-2 inhibition for
breast cancer prevention. Limited data are
available in animal models of breast cancer;
however, Harris et al. (426) showed that cele-
coxib inhibited the tumor multiplicity by 86%
and the incidence by 68%.
Clinically, Celebrex has been shown to be
effective in FAP patients. Steinbach et al.
(424) showed that twice-daily 400 mglday of
Celebrex reduced the number of polyps in fa-
milial adenomatous patients by 28% after 6
months of treatment. There was a similar in-
cidence of adverse events in all of the treat-
ment groups. These data were sufficient for
Celebrex to be approved for use in FAP.
Rofecoxib was shown to be effective in che-
moprevention of polyps in the APC delta 716
knockout mice by Oshima et al. (428). Rofe-
coxib was given in food for 8 weeks and then
the animals were necropsied and the polyps
scored. There was a greater than 57% inhibi-
tion of polyps greater than 1mm in size and up
to 100% inhibition of polyps greater than 3
mm. Overall, the data are very interesting and
suggestive of potential activity for Vioxx in
FAP, although thus far no clinical data have
been reported.
250
12 CONCLUSION A N D SUMMARY
The modulation of the synthesis and activity
of a number of eicosanoid ~roductshas been
A
shown to be of significant importance medi-
cally in a multitude of clinical conditions. Non-
selective cyclooxygenase inhibitors (NSAIDs)
have been broadly used for several decades in
the relief of pain and inflammation. The new
COX-2-selective agents (Table 5.2) have al-
ready proved to be safer and equally effica-
cious. It is likely that they will continue to
replace the older agents. However, the cardio-
vascular effects of COX-2 agents will surely
need to be watched carefully in the future. In
addition, the safety profile of these agents will
allow for their use in many clinical settings,
where the possibility of GI bleeding or platelet
effects preclude the use of NSAIDs. These
could include cancer isrevention, cancer ther-
apy, and both surgical and cancer pain.
Blockade of the effect or synthesis of leuko-
trienes has proved to be a safe and effective
approach to asthma therapy (Table 5.2). Here
again, especially for the leukotriene biosyn-
thesis inhibitors, new uses in other pulmonary
diseases are likely. Expansion into other aller-
gic diseases is already taking place and will
probably continue.
In addition to the already established ap-
proaches such as COX-2 inhibitors, 5-LO in-
hibitors, and CYSLTl antagonists, several
new targets in eicosanoid research are clearly
not well explored and have significant poten-
tial. These would include inhibitors of LTA,
hydrolase, LTB, antagonists at both recep-
tors, CysLT2 antagonists, inhibitors of induc-
ible PGE, synthase, selective prostaglandin
receptor antagonists, lipoxin modulators, and
perhaps COX-3 inhibitors (D. Simmers, sub-
mitted, 2002). Whatever the final success of
any one of these targets, it is clear that modu-
lation of eicosanoid production and action pro-
vides clear medical benefit in multiple set-
tings.
13 ABBREVIATIONS
COX cyclooxygenase
DP dipeptidase
FLAP 5-LO activating protein
COX-2 inhibitors and Leukotriene Modulators
gGTP gamma glutamyl transpeptidase
HETE hydroxyeicosatetraenoic acid
HPETE hydroperoxyeicostetraenoicacid
LO lipoxygenase
LT leukotriene
PG prostaglandin
PLA phospholipase
TX thromboxane
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CHAPTER SIX

Agents Acting on Prostanoid

Receptors
DAVIDW. JENKINS
PATRICK P.A. HUMPHREY
University of Cambridge
)GlaxoInstitute of Applied Pharmacology
$Cambridge,United Kingdom

I
BERT A. COLEMAN
agene Laboratories Ltd.
!byston, United Kingdom

Contents
1 Introduction, 266
1.1 Historical Background and Overview of
Prostanoid Synthesis, 266
1.2 Biological and Therapeutic Functions of
Prostanoids, 267
1.3 Prostanoid Receptor Classification: A
Historical Perspective, 269
2 Natural Prostanoids, 269
2.1 Biosynthesis, 269
2.2 Catabolism, 270
2.3 Physiological Actions of Prostanoids, 271
2.3.1 Prostaglandin D,, 271
2.3.2 Prostaglandin E,, 272
2.3.3 Prostaglandin F,,, 274
2.3.4 Prostaglandin I,, 274
2.3.5 Thromboxane 4 , 2 7 5
3 Prostanoid Receptor Classification and
Characterization, 275
3.1 Prostanoid Receptor Subtypes and Isoforms,
276
3.2 Prostanoid Receptors as G-protein-Coupled
Receptors, 277
3.3 Radioligand Binding Studies, 278
3.3.1 PGD,-Specific Binding Sites, 278
3.3.2 PGE,-Specific Binding Sites, 278
er's Medicinal Chemistry and Drug Discovery 3.3.3 PGF,,-Specific Binding Sites, 280
Edition, Volume 4: Autocoids, Diagnostics, 3.3.4 PG12-SpecificBinding Sites, 280
m New Biology 3.3.5 TXA&3pecificBinding Sites, 280
nald J. Abraham 3.3.6 Summary, 280
N 0-471-37030-4 O 2003 John Wiley & Sons, Inc. 3.4 Signal Transduction, 281
 Prostanoid

AVID W. JENKINS

BERT A. COLEMAN

ston, United Kingdom

Contents
1 Introduction, 266
1.1 Historical Background and Overview of
Prostanoid Synthesis, 266
1.2 Biological and Therapeutic Functions of
Prostanoids, 267
1.3 Prostanoid Receptor Classification: A
Historical Perspective, 269
2 Natural Prostanoids, 269
2.1 Biosynthesis, 269
2.2 Catabolism, 270
2.3 Physiological Actions of Prostanoids, 271
2.3.1 Prostaglandin D,, 271
2.3.2 Prostaglandin E,, 272
2.3.3 Prostaglandin F,,, 274
2.3.4 Prostaglandin I,, 274
2.3.5 Thromboxane 4 , 2 7 5
3 Prostanoid Receptor Classification and
Characterization, 275
3.1 Prostanoid Receptor Subtypes and Isoforms,
276
3.2 Prostanoid Receptors as G-protein-Coupled
Receptors, 277
3.3 Radioligand Binding Studies, 278
3.3.1 PGD,-Specific Binding Sites, 278
3.3.2 PGE,-Specific Binding Sites, 278
r's Medicinal Chemistry and Drug Discovery 3.3.3 PGF,,-Specific Binding Sites, 280
Edition, Volume 4: Autocoids, Diagnostics, 3.3.4 PG1,-Specific Binding Sites, 280
Drugs from New Biology 3.3.5 TXA&3pecific Binding Sites, 280
by Donald J. Abraham 3.3.6 Summary, 280
-471-37030-4 0 2003John Wiley & Sons,Inc. 3.4 Signal Transduction, 281
265

3.4.1 DP Receptors, 281
3.4.2 EP, Receptors, 281
3.4.3 EP, Receptors, 281
3.4.4 EP, Receptors, 281
3.4.5 EP, Receptors, 281
3.4.6 FP Receptors, 281
3.4.7 IP Receptors, 282
3.4.8 TP Receptors, 282
4 Prostanoid Receptor-Selective Ligands and
Structure-Activity Relationships, 282
4.1 DP Receptors and Selective Ligands, 282
4.2 EP Receptors and Selective Ligands, 285
4.2.1 General Considerations, 285
4.2.2 EP, Receptor-Selective Ligands, 286
4.2.3 EP, Receptor-Selective Ligands, 287
4.2.4 EP, Receptor-Selective Ligands, 288
4.2.5EP, Receptor-Selective Ligands, 289
4.3 FP Receptors and Selective Ligands, 289
4.4 IP Receptors and Selective Ligands, 292
1 INTRODUCTION
1.1 Historical Background and Overview
of Prostanoid Synthesis
Prostanoids are metabolites of C,, fatty acids
that act as local hormones in both the periph-
ery and central nervous system (CNS). Classi-
cally, they are comprised of the prostaglandins
and thromboxanes. However, recently, inter-
est has focused on the isoprostanes, which are
formed through the direct oxidation of mem-
brane phospholipids and not through the ac-
tions of cyclooxygenase (COX) enzymes. De-
spite this difference in synthesis, current
evidence suggests that they act predominantly
through prostanoid receptors (1). Another in-
teresting recent development has been the dis-
covery of the prostamides, which are deriva-
tives of anandamide and are formed through
the actions of COX-2 and not COX-1 (2-4).
As a family, the prostanoids are almost
ubiquitously distributed, and in general, have
a short duration of action. Von Euler first pro-
posed the term 'prostaglandin' in describing
the presence of a vasodepressor and smooth
muscle stimulating factor in human seminal
fluid (51, following the initial observations of
Kurzrok and Lieb (6) and Goldblatt (7). How-
ever, it was another 20 years before the first
prostaglandins, prostaglandin El(PGE,) and
prostaglandin F,, (PGF,,), were successfully
purified (8). During the next decade it became
Agents Acting on Prostanoid Receptors
4.5 TP Receptors and Selective Ligands, 294
4.6 Prostanoid Receptors: Location of Ligand
Binding Sites, 295
5 Clinical Use of Agents, 298
5.1 Natural Prostaglandins, 299
5.1.1 Alprostadil (Prostaglandin El),
299
5.1.2 Dinoprostone (ProstaglandinE,), 301
5.1.3 Dinoprost (Prostaglandin F,,), 302
5.1.4 Epoprostenol (Prostaglandin I,;
Prostacyclin), 302
5.2 Synthetic Prostaglandins, 303
5.2.1 Gemeprost, 303
5.2.2 Misoprostol, 303
5.2.3 Sulprostone, 304
5.2.4 Latanoprost, 304
5.2.5 Iloprost, 304
5.2.6 Carboprost, 305
5.2.7 Limaprost, 305
6 New Developments, 305
apparent that the family included more than
the original two and these were named alpha-
betically from PGA, to PGH,. Of these, PGG,
and PGH, are unstable intermediates (9) gen-
erated during the biosynthesis of this family
of hormones. In the mid-1970s, two new pros-
taglandin-like compounds were discovered:
thromboxane A, ( T W ) (10) and prostacyclin
(PGI,) (11).
The natural prostanoids derived from ara-
chidonic acid are known as prostaglandin D,
(PGD,), prostaglandin E, (PGE,), prostaglan-
din F,, (PGF,,), prostaglandin I, (PGI,), and
thromboxane A, (TXA,). They exert their
physiological and pathophysiological actions
through specific high affinity receptors and
are responsible for a multitude of actions,
some of which have been exploited to produce
clinically effective medicines. Prostaglandins
are characterized by a cyclopentane ring, with
two side-chains, termed CY and w, that are both
in the trans configuration. Also present are a
hydroxyl group at C-15 and a A13 trans double
bond. The structure of PGE, as representative
of the entire group is shown in Fig. 6.1. Pro-
stanoids can be biosynthesized from three re-
lated fatty acid precursors to give rise to three
different series based on the number of double
bonds in their side chains. Thus, prostanoids
derived from 8,11,14-eicosatrienoic acid (y-
homolinolenic acid) give rise to the 1-series,
whereas the 2- and 3-series are derived from
1 Introduction
Cyclopentane ring
a-chain
Hydroxy group at C-15
trans doubli bond
Figure 6.1. The structure of prostaglandin E,.
Note the presence of the a- and o-chains, the cyclo-
pentane ring, trans double bond, and hydroxy group
at C-15.
5,8,11,14-eicosatetrienoicacid (arachidonic
acid) and 5,8,11,14,17-eicosapentaenoicacid
(timodonic acid), respectively. Arachidonic
acid is the most abundant precursor in mam-
mals, including humans, making the 2-series
by far the most common. However, eicosapen-
tanoic acid (EPA, 20:5w3) is present in large
amounts in marine animals, and consequently
in the bodies of those species living largely on
a diet of these animals. For example, Green-
land Eskimos have much higher levels of EPA
and other 0 3 fatty acids in their phospholip-
ids, and this has been linked to the lowered
incidence of heart disease in these populations
(12).
Prostaglandin H, is the common biosyn-
thetic precursor of the natural prostanoids,
and is produced as a result of the action of
cycloxygenases on free arachidonic acid. The
major source of free arachidonic acid is the
phospholipids of the cell membrane, from
which it is largely liberated by the actions of
phospholipases. The activity of COX, also
known as prostaglandin H synthase (PGHS),
converts arachidonic acid to PGH, through a
two-step process: first a cyclooxygenase step,
that results in the production of the unstable
intermediate PGG,, and second, a peroxidase
step that yields PGH,, which is then converted
to the individual prostanoids by specific syn-
thases. An overview of this process is shown in
Fig. 6.2. The biosynthesis and breakdown of
prostanoids is discussed further in Sections
2.1 and 2.2.
1.2 Biological and Therapeutic Functions
of Prostanoids
The major roles of prostanoids are in the au-
tocrine and paracrine control of physiological
processes, and therapeutically they have
found use mainly in obstetrics, gastoenterol-
ogy, ophthalmology, and the treatment of car-
diovascular disease. In the reproductive sys-
tem, prostaglandins, especially of the E and F
series, have potent effects on uterine contrac-
tion and have been used to stimulate abortion,
cervical ripening, and the induction of labor
(13). In addition, in many laboratory and farm
animals, but not in humans, PGF,, is a potent
luteolytic agent and is used for this purpose in
veterinary medicine (14). Similarly, prosta-
glandin receptors exhibit a widespread distri-
bution in the gastrointestinal tract (15), and
prostaglandins are generally thought to have
cytoprotective effects in the gastric mucosa
(16), through the control of gastric secretions,
protection of the mucosal barrier, and the reg-
ulation of blood flow (17). This assertion has
led to the production of a number of PGE an-
alogs for use as potential gastroduodenal pro-
tective agents, against, for example, the ef-
fects of non-steroidal anti-inflammatory drugs
(NSAIDs). Recently, PGF analogs, such as la-
tanoprost, have found use as novel medicines
for the treatment of glaucoma (18). In the car-
diovascular system, prostanoids, especially
prostacyclin, are involved in, for example, the
regulation of platelet aggregation. Prostacy-
clin and its analogs are administered exog-
enously in the treatment of several forms of
cardiovascular disease, where they may cause
vasodilatation and also inhibit platelet clump-
ing and aggregation (19). Prostaglandins are
also active in the renal system. Indeed, one of
the earliest physiological effects attributed to
prostaglandins was the effect of PGE, to block
the action of vasopressin on renal water per-
meability (20). Receptors for prostaglandins
have also been localized to all parts of the kid-
ney (21). Prostaglandins, particularly of the E
and I series, have also been strongly impli-
cated in inflammation and inflammatory pain
(22), leading to recent efforts to produce spe-
cific receptor antagonists for the treatment of
these conditions.
 Agents Acting on Prostanoid Receptors

Arachidonic acid

I cyclooxygenase

Prostaglandin
H2 synthase 0'
o H OOH
Prostaglandin G2

Peroxidase

'\_/V\CO~H
M..
HO.

0
'
+\-CO~H
PGD s y n t h y

OH
0'
0 H
I
Xvvvv
O

PGE synthase
, ,
/
Ysynthas
/ .
H
Prostaglandin H2

PGF synthase
\
'"\---=/\/\C O ~ H
/ .
OH
Prostaglandin D2
0
I!
I
PGI "q
\ Thromboxane A2

. A \ \ = / \ / \ ~ ~ ~ ~ synthase -\\I. CO~H

HO OH HO OH
Prostaglandin Prostaglandin F2,

Prostaglandin l2

Figure 6.2. The biosynthesis of prostanoids from arachidonic acid. Free arachidonic acid is con-
verted to the unstable intermediates PGG, and PGH, by cyclooxygenase (COX) enzymes. PGH, is
then converted to the five primary prostanoids by specific synthases.
2 Natural Prostanoids
1.3 Prostanoid Receptor Classification:
A Historical Perspective
Prostanoids, as metabolites of fatty acids, are
relatively hydrophobic compounds and it was
initially thought that they might act through
incorporation into the plasma membrane.
However, it has subsequently been shown that
prostanoids predominantly exert their biolog-
ical actions through interactions with specific
receptors in cell membranes. In the absence of
direct evidence, this conclusion is suggested
by a consideration of some of the basic proper-
ties and actions of the prostanoids. For exam-
ple, they are active at low concentrations
(<W9 MI,and thus display high potency, im-
plying the existence of specific binding sites.
Furthermore, it is clear that different mem-
bers of the prostanoid family can evoke differ-
ent responses in the same system. For exam-
ple, TXA, and PGI, exert both aggregatory
and non-aggregatory effects on human plate-
lets, respectively, thus suggesting, in platelets
at least, the presence of distinct receptor sites
for both of these prostanoids.
The first evidence for the existence of more
an a single subtype of prostanoid receptor
e from the work of Pickles (23), who used
0th natural and synthetic analogs of E and F
ries prostaglandins to demonstrate differen-
a1effects in several smooth muscle prepara-
ons: the rabbit jejunum and human and
inea pig myometrium. These studies led
m to conclude that there were at least three
es of prostanoid receptors. The presence of
ultiple receptors was also suggested by the
ork of Andersen and Ramwell (241,who com-
d the agonist potency ratios of PGA, PGE,
PGF analogs, and Gardiner and Collier
who examined the prostanoid receptor
s present in the lung. In 1982, Kennedy
co-workers proposed the first overall clas-
%cation of prostanoid receptors. In the pre-
olecular biology era, they proposed specific
receptors for PGD,, PGE,, PGF,,, PGI,, and
%, calling them DP, EP, FP, IP, and TP
receptors, respectively. At each of these recep-
tors, one of the natural prostanoids was at
east one order of magnitude more potent than
the other four. Hence, PGD, is the most po-
tent natural agonist at the DP receptor, PGE,
the most potent natural EP receptor agonist
and so on. This system was based on func-
tional data obtained with the natural agonists,
some synthetic agonists, and a small number
of antagonists, but is still valid today (1,26).
These initial studies were conducted on iso-
lated smooth muscle preparations that were
considered PGE- (e.g. guinea pig ileum), PGF-
(e.g. dog and cat iris), or TXA-sensitive (e.g.
rat aorta). They compared the agonist poten-
cies of the naturally occurring prostanoids and
the TXA, mimetic, U46619, in these prepara-
tions. Coupled with other studies that sug-
gested receptors for PGD, and PGI,, this led
to the proposal outlined above that receptors
existed for each natural prostanoid. Further
studies identified compounds that acted as re-
ceptor blockers in some preparations, but not
others. Hence, AH19437 was identified as a
competitive antagonist in TP receptor con-
taining tissues (26). Whilst SC-19220 was an
antagonist in some PGE-sensitive tissues (261,
producing rightward shifts in the concentra-
tion-effect curves in guinea pig ileum and
guinea pig and dog fundus, it had negligible
effects in the PGE-sensitive preparations cat
trachea or the chick ileum (26). This provided
the first evidence for a subdivision of the EP
receptors. Further evidence came from the
identification and use of the antagonist,
AH6809, and the agonists, sulprostone and
AY23626, with AH6809 being active in EP,
receptor-containing preparations, and sulpr-
ostone and AY23626 having actions at EP,
and EP,, and EP, and EP, receptors, respec-
tively (27). Subsequently, a fourth EP recep-
tor was identified in piglet saphenous vein (28,
29). The rank order of the naturally occurring
prostaglandins at their receptors is shown in
Table 6.1.
This chapter will provide an overview of the
natural prostanoids and their synthetic and
metabolic pathways and then discuss the recep-
tors at which prostanoids exert their actions and
the agents that have been generated to ad as
either specific receptor agonists or antagonists,
some of which have found use in the clinic.
2 NATURAL PROSTANOIDS
2.1 Biosynthesis
Prostanoids are synthesized de novo by a wide
variety of cells, and synthesis can be induced
270
Table 6.1 Rank Orders of Potency of the Naturally Occurring Prostanoids for Each of the
Five Types of Prostanoid Receptors
Receptor Type Subtype
DP DP1
EP EPl
EP2
EP3
EP4
FP
IP IPI
TP
"U46619 is a stable TXA, mimetic (see Section 4.5) and was used instead of TXA,. Data taken from Coleman (1).
by a variety of stimuli, including non-prostan-
oid receptor activation as well as simple me-
chanical agitation. Prostanoid synthesis re-
quires the presence of free, unesterified
arachidonic acid. Under resting conditions,
the cytoplasmic levels of this fatty acid are
low, necessitating the liberation of this sub-
strate as the first and rate-limiting step in pro-
stanoid biosynthesis. The main source of ara-
chidonic acid is that present in membrane
glycophospholipids, esterified in the 2-acyl po-
sition in the fatty acyl chains, and this is pri-
marily liberated by the enzyme phospholipase
A, in a one-step process (30). Arachidonic acid
not metabolized by cyclooxygenases or other
eicosanoid-producing enzymes (e.g., lipoxyge-
nases) is rapidly re-esterified by acetyltrans-
ferases (31).
Arachidonic acid, liberated from mem-
brane phospholipids, can be metabolized to
the five primary prostanoids, PGD,, PGE,,
PGF,,, PGI,, and T-. The first two steps in
this process are catalyzed by the cyclooxygen-
ase (COX) enzymes and thereafter by specific
prostanoid synthases (32). An overview of pro-
stanoid synthesis is illustrated in Fig. 6.2.
2.2 Catabolism
The half-life of most prostaglandins in the cir-
culation is less than 1 min, and this is attrib-
utable to the efficiency of the processes that
exist to aid in their inactivation. For example,
in the lung, around 95% of single doses of ei-
ther PGE,, PGE,, or PGF,, are metabolized
during the first passage. The enzymes in-
volved in prostanoid metabolism are located
intracellularly, thus necessitating the uptake
of substrates across the cell membrane (33-
Agents Acting on Prostanoid Receptors
Rank Order of Natural PGsa
D, > E, = F,, = I, = U46619
E, > F,, = I, > D, > U46619
E, > F,, = D, = I, > U46619
E, > I, > F,, >> D, > U46619
E, >> I, = F,, > D, > U46619
F,, > D, > E2 = U46619 > 1,
I, > D, = E, = F,, > U46619
U46619 >> D, = E, = F,, = I,
36). This is believed to be a carrier-specific
mechanism and displays differential selectiv-
ity over the prostanoid-catabolizing enzymes.
Following uptake, prostanoid metabolism oc-
curs in two stages: first, a rapid inactivation by
prostanoid-specific enzymes. This first stage
normally results in a loss of biological activity.
Second, a slower inactivation process occurs,
involving enzymes that are responsible for
general fatty acid oxidation. Metabolism oc-
curs in the lung and also the liver and the
kidney.
The first important work on prostanoid
metabolism was conducted by Anggard and
Samuelsson (37),who demonstrated that PGE
and PGF compounds are dehydrogenated at
carbon-15 to the corresponding 15-keto pro-
stanoid by the nicotinamide adenine dinucle-
otide + (NAD+ )/nicotinamide adenine dinu-
cleotide phosphate (NADP)-linked enzyme,
15-hydroxy-prostaglandin dehydrogenase
(PGDH), to form 15-keto-PGE, and 15-keto-
PGF,,. PGDH has subsequently been shown
to have a distribution pattern that includes
the lung, kidney, and liver (38). Several com-
pounds can inhibit PGDH, including the sub-
stituted phenylazobenzoic acetic acid deriva-
tives, Ph CL28A and Ph CK61A (39). PDGH is
not specific to prostaglandins. For example,
the NAD+-linked enzyme present in the lung
will also metabolize 15-hydroxyeicosatet-
reonic acid (15-HETE) and other o6-contain-
ing fatty acids (40). These 15-keto metabolites
are far less active than the parent compounds,
and therefore, these initial transformations
represent a functional inactivation.
The second stage of inactivation is cata-
lyzed by a A13,14 reductase and involves satu-
2 Natural Prostanoids
ration of the A13 double bond to form 15-keto,
13,14-dihydro prostaglandins. This reaction
can only occur in prostaglandins that have
been initially undergone oxidation of the 15-
hydroxy group, and the metabolite formed is
the major breakdown product found in plasma
samples and kidney and lung homogenates.
This enzyme uses nicotinamide adenine dinu-
cleotide phosphate hydrogen (NADPH) as a
cofactor and can be inhibited by p-chloromer-
curibenzoic acid (41).
Following these steps, /3 and w oxidation
take place to shorten the carboxyl side-chain
and to introduce a further carboxyl group, re-
spectively (42-45). The urinary metabolites
detected~after prostaglandins are infused in
man are generally dinor and tetranor com-
pounds, so the carboxyl-containingside-chain
is normally shortened by two to four carbon
atoms. This is the result of p-oxidation that
occurs mainly in liver mitochondria or peroxi-
somes. The w-oxidation that occurs is catal-
ysed by cytochrome P450, and occurs largely
in the liver and kidney, and many of the uri-
nary metabolites of prostaglandins are gener-
ated through this pathway. Overall, these
: steps help to make the prostanoids more polar
I and thus available for excretion by the kidney.
j Thus, the final metabolites found in the urine
are 16-18 carbon atoms and possess carboxyl
oups on both the a and w side-chains.
Prostaglandin D, follows the same basic
ples outlined above, but in humans at
t, a different pattern of catabolism may
cur. It has been found that a sizeable pro-
rtion of urinary metabolites obtained fol-
lowing injection are recovered as metabolites
of PGF compounds (46). For this to occur, the
11-ketogroup of PGD, must be reduced, but it
is unclear whether this reduction is of PGD,
itself, or of one of its subsequent metabolites,
for example, those that are present after being
oxidized by PGDH.
Prostacyclin, although not taken up by the
lungs, is rapidly inactivated by acid-catalysed,
non-enzymatic means to the metabolite 6-
keto-PGF,,. In addition it is also catabolized
by PGDH to 6,15-diketo metabolites. 6-keto-
PGF,, is biologically inactive and also rapidly
metabolised in vivo, with the major urinary
oduct being dinor-6-keto-PGF,, (47).
Thromboxane A, has a plasma half-life as
short as 7 s (48) and spontaneously decom-
poses non-enzymatically to the functionally
inactive TXB,, which is either excreted unal-
tered or as one of several other metabolites.
For example, TXB, can undergo p-oxidation
to dinor-TXB,.
2.3 Physiological Actions of Prostanoids
Prostaglandins have a wide range of actions in
normal cell physiology, but also as pathophys-
iological agents in, for example, inflammation
and pain states. The roles of the natural pro-
stanoids are reviewed in this section in rela-
tion to studies on receptor distribution, which
provide useful information concerning poten-
tial pharmacological applications. Recent ad-
vances in molecular biology have also allowed
the creation of prostanoid receptor "knock-
out" mice, which have proved valuable in help-
ing to further elucidate the receptor types in-
volved in mediating the various actions of
prostanoids.
2.3.1 Prostaglandin D,. PGD, acts pre-
dominantly at DP receptors, although it does
have relatively high potency at FP and even
TP receptors. DP receptors are probably the
least widely distributed of all the prostanoid
receptors and often co-exist with other prosta-
noid receptors, making them difficult to study
outside of recombinant systems. However, the
availability of potent, selective agonists and
antagonists (see Section 4) has proved pivotal
in surmounting this problem.
DP receptors are present on vascular
smooth muscle, platelets, and in nervous tis-
sue, including the central nervous system
(CNS). In the CNS, PGD, has been associated
with sleep induction, as well as body tempera-
ture regulation, hormone release, nociception,
and olfactory function. DP receptors have also
been localized to smooth muscle in the gut,
uterus, and airways. Northern blot and in situ
hybridization studies have reaffirmed this,
with only low levels of expression being de-
tected in human retina and small intestine
(49). The physiological actions of PGD,, medi-
ated through DP receptors, are generally "in-
hibitory"; that is, smooth muscle relaxation
and the inhibition of platelet aggregation.
However, "excitatory" actions have also been

reported: for example, in afferent sensory
nerves PGD, seems to induce hyperalgesia
(50) and can also stimulate the release of neu-
ropeptides (51). PGD, has also been impli-
cated in central roles, such as sleep-induction,
in both rats and humans (52). DP receptors
have been localized to the leptomeninges (53),
and PGD, injected into the arachnoid space
below the basal forebrain caused leptomenin-
geal cellular activation, detected by immuno-
histochemistry for the immediate early gene,
fos (54). Furthermore, the brain-specific iso-
form of PGD synthase has recently been local-
ized in the leptomeninges (55). DP receptors
have also been recently detected in the mu-
cous-secreting goblet cells in the rat gastroin-
testinal tract, suggesting a potentially novel
role for PGD, in mucous secretion (56).
DP receptor localization also seems to vary
considerably between species. For example,
platelet DP receptors seem to be most abun-
dant in humans and not in other animal spe-
cies (57). PGD, is also the major prostaglandin
produced by mast cells following immunologi-
cal challenge (58). Recent studies using mice
deficient in the DP receptor have suggested
that PGD, production may be important in
some aspects of asthma (59). In a model of
asthma, when challenged with ovalbumin,
DP-'- mice showed reduced lymphocyte accu-
mulation in the lung compared with wild-type
controls, and in addition, failed to develop air-
way hyperreactivity. These data suggest that,
in mice at least, DP receptors may play a role
in airway hyperreactivity.
2.3.2 Prostaglandin E,. PGE, acts predom-
inantly through EP receptors, which are
abundantly distributed and are responsible
for mediating the multitude of actions of
PGE,. These include smooth muscle contrac-
tion and relaxation, stimulation and presyn-
aptic inhibition of neurotransmitter release,
neuronal sensitization, nociception, inhibition
of lipolysis, and gastric acid secretion and
modulation (positively and negatively) of wa-
ter secretion. It was partly because of the
sheer diversity of these responses and through
the use of what are now known to be subtype
specific ligands that it became clear that there
was more than one subtype of EP receptor. It
Agents Acting on Prostanoid Receptors
is now known that at least four receptor sub-
types exist, and these are termed EP,, EP,,
EP,, and EP, receptors (see Section 3).
The first characterized EP receptor sub-
type, the EP, receptor, is probably the least
widely distributed of all the EP receptors. It
predominantly mediates the smooth muscle
contraction actions of PGE, and is present in
guinea pig trachea, gastrointestinal tract,
bladder, and uterus. This receptor subtype has
also been found in other animal species: for
example, in the bovine iris sphincter (60), dog
gastric fundus (61), and human myometrium
(62). These receptors also seem to play a role
in inflammatory pain, with EP, antagonists
currently being investigated as potential an-
tinociceptive agents (63).In situ hybridization
studies have detected EP, receptor transcripts
in the kidney, lung, and stomach of the mouse
(64, 65). Recently, EP, receptor knockout
mice have been used to suggest that endoge-
nous EP, receptors are important in the me-
diation of pain perception and also the regula-
tion of blood pressure (66). EP, receptor-
deficient mice were both healthy and fertile
but showed reduced pain sensitivity, similar to
that obtained following inhibition of COX
with an NSAID. Similar results have also been
reported in mice deficient in the IP receptor
(67)(see Section 2.3.4), thus suggesting that
EP, and IP receptors may represent possible
novel targets for the treatment of inflamma-
tory pain.
EP, receptors, first recognized because of
the lack of effect of EP, receptor antagonists
in blocking the relaxant effects of PGE,, have
a more widespread distribution than the EP,
receptor subtype. This receptor subtype also
mediates a more diverse array of actions. For
example, smooth muscle EP, receptors always
mediate smooth muscle relaxation [through
increases in cyclic adenosine monophosphate
(CAMP)through G, (see Section 3.3)]. Epithe-
lial EP, receptors regulate non-acid secretion,
and in mast cells and basophils, they mediate
inhibition of mediator release. Furthermore,
they may also mediate afferent sensory nerve
activation, including neuropeptide release.
Northern blotting in the mouse has suggested
the presence of EP, receptor mRNA in the il-
eum, thymus, spleen, heart, and uterus (68).
This receptor subtype has also been localized
 Figure 2.8. JOM-13 (blue) in the 8-opioid receptor binding pocket (stereoview). [Taken from Fig.
2.9 in H. I. Mosberg, Bwpolymers (Peptide Science), 51, 426 (1999). Reprinted by permission of
John Wiley & Sons.]
-

Figure 7.15. Rhcuk~psincrystal stpcture. Thedimensional structure of rhodopsin based on X-ray

crystallography (186). Note that all-trans-retinal is protected from the intradiscal side by multiple
' structural elements, including several f3 strahds. Carbohydrates are in blue, 11-cis-retinal is in
green, and helices are-in gray. Red sh;ldows are added for esthetic reasons. This figure was gener-
ated by C. Behnke (University of Wa~hingto~n) and is reprinted with permission from Prog. Retin.
Eve Res., 20,469-521 (2001).
 (a)
Figure 7.21. Schematic represen- h
tation of DNA binding domains. RW
Structures of DNA-binding com-
plexes involving RXR and their
dimer interfaces. The overall struc-
tures are shown on the left, with
close-up views of the protein-pro-
tein interactions shown on the
right. Dotted blue lines indicate
hydrogen bonds between proteins
or between proteins and the DNA
spacing. The dotted surface indi- (c)
cates complementary van der Wads
interactions. DNA sequences (cyan)
are shown with their 5' ends point- RXR
ing up, with base pairs belonging
to the spacing element of the DRs
shown schematically in red. In
each case, protein-protein contacts
are formed directly over the minor
groove of the spacing, with several
protein-DNA phosphate contacts
stabilizing the assembly. The inter-
acting amino acid side-chains are
shown in green, with nitrogen
atoms indicated in blue and oxygen
atoms indicated in red. (a and b) RAR ,
The RXR-TR DBD heterodimeric
complex with D w (c and d) the
RXR DBD homodimeric complex
with DR1, and (e and 0 the RXR-
RAR DBD heterodimeric complex
with DR1. Reprinted with permis-
sion from Cum Opin. Struct. Biol.,
11,3338 (2001).

Figure 7.22. Schematic representa-

tion of apo- and holo-RXR ligand bind-
ing domain. Helices 1-12 (Hl-H12)
are indicated. Helices indicated in yel-
low and red represents the apo- and
holo-forms of the receptor, respective-
ly, whereas helices indicated in blue
and green are positioned similarly in
both forms of the receptor. The arrows
represent movement of the helices to
accommodate 9-cis-RA in the binding
pocket (indicated). This figure was
kindly supplied by Dr. Pascal Egea.
 Photolithograph
mask

Figure 11.2. Photolithographic process for on-chip synthesis of oligonucleotides. A: the steps in
this process in two cycles of nucleotide addition. A lithographic mask and light source are shown
panel a that results in exposure of specific spots on the microarray chip. The light activates
ctive groups on the chip such that nucleotide coupling can occur only on the specific spots as
n in panel b. The nucleotide added is shown by green blocks and is thymidine for this exam-
ure of new spots to light in a second round of light
next round of nucleotide addition in which the red
ple adenosine. This process is repeated until all

11.3. Hybridization of two differentially
am shows a single array spot with probe
strands (in gray) bound to the chip. cDNA
led with Cy3 (green) and one labeled with Cy5
1, and hybridized to the spotted DNA.
Figure 11.4. A spotted DNA array with two-color
detection of hybridization. An example of a spotted
DNA array (16 X 20 elements) is shown after
hybridizaton with two differentially labeled cDNA
preparations, Cy3 (pseudo-colored green) and Cy5
(pseudo-colored red). The overlaying of the green
and red images produces the image shown. The
hue of each spot, ranging from green to red, indi-
cates the relative expression level for the gene
specific for each spot (image courtesy Packard
Biochip Technologies).
 BRE MDA-MB-435
BRE MDA-N -
I
MEL
MEL
MEL
718 Melanomas MEL
MEL
MEL
MEL
CNS
CNS
BRE
CNS
616 CNS Cancers CNS
CNS
BRE
CNS SF-295 I
NSC HOP-92

B
BRE MDA-MB-231
BRE ADR RES
OVA OVCAR-8
NSC HOP-62
REN SNl2C
REN
REN
REN
Renal Cancers REN
REN ACHN
REN CAKI-I
REN A498
MEL LOXlMVl I
LEU CCRF-CEM
LEU MOLT-4
LEU HL-60
616 Leukemias LEU SR
LEU RPMI-8226
LEU
OVA
OVA
OVA
PRO

-
PRO
OVA
OVA
BRE
BRE
COL
COL
COL
Colon Cancers COL
COL
COL
COL
NSC

519 Lung Cancers

NSC A549
EKVX
-NSC
NSC
Figure 11.5. Cluster analysis of the NCI 60 cell lines. Gene expression data from the Ross et al.
(12) study was filtered to contain only genes with complete data for all cell lines and genes with
the highest variance across cell lines. Only 548 genes were selected for this analysis based on
genes with variance of greater than twice the variance for the entire dataset. The cluster distance
or similarity calculation was based on the Pearson correlation coefficient, and clusters were linked
by average linkage using the software SPSS. Most cell lines can be seen to cluster based on tissue
of origin, shown by the labels on the left and the fraction of the total number of cell lines for each
tissue. The abbreviations for tissue types are a s follows: BRE, breast cancer; CNS, central nerv-
ous system cancers; COL, colon cancers; LEU, leukemias; MEL, melanomas; NSC, non-small cell
lung cancers; OVA, ovarian cancers; PRO, prostate cancers; REN, renal cancers.
2 Natural Prostanoids
to mouse glomeruli of the kidney (65). Fur-
thermore, it seems that this receptor may be
upregulated in response to cell stimulation.
This has been demonstrated in a macrophage
cell line in response to lipopolysaccharide (69).
EP, receptor knockout mice have also been
used to further highlight the functions of
PGE, in the cardiovascular system. For exam-
ple, Kennedy and colleagues (70) examined
the effects of PGE, and receptor selective an-
alogs in both wild-type and EP,-'- mice. In
wild-type animals, the EP, receptor selective
agonist, butaprost, evoked a transient hypo-
tension, which was not observed in mice defi-
ent in the EP, receptor. However, the hyper-
nsive effects of sulprostone were similar in
th groups of animals. Hence, the EP, recep-
may be important, in the mouse vascula-
at least, in mediating some of the vasodi-
ory responses to PGE,.
EP, receptors are probably the best studied
all the EP receptors. They are present in
trointestinal, uterine, and vascular smooth
le, where they cause smooth muscle con-
ion. In autonomic nerves they cause inhi-
ion of neurotransmitter release, and in adi-
es, they inhibit lipolysis (71). In gastric
sal cells, they inhibit gastric acid secre-
n (721, and E series prostaglandins are well-
acterized stimulators of mucus and bicar-
ate secretion. Several PGE analogs have
n developed to exploit this role as it was
thought that EP, receptor agonists might be
useful in the treatment of gastric ulceration
d also prophylaxis of the side effects associ-
th long-term use of aspirin and other
s (see Section 4.2.4). In renal medulla
regulate the inhibition of water reabsorp-
(73).Distribution studies using Northern
ting and in situ hybridization reveal
A expression in the kidney and uterus, as
as the stomach, spleen, brain, and lung
. Furthermore, this receptor subtype has
localized to the tubules in the medulla
so the macula densa and distal tubules
n the kidney itself (75). The receptor dis-
ution has also been examined in neuronal
ue, and has been reported in both dorsal
ganglion and trigeminal ganglion tissue
also within the brain in areas including
hypothalamus, hippocampus, locus coer-
s, and raphe nuclei (75, 76). Within the
hypothalamus, EP, receptor transcripts have
been detected in cells surrounding the orga-
num vasculosa lamina terminalis (OVLT),and
EP, receptor activation here has been associ-
ated with fever generation. E series prosta-
glandins are potent fever inducers when in-
jected into the brain, and their involvement in
the central mediation of fever was proposed as
early as 1970 (77). At the spinal cord level, it
has been suggested that PGE, may induce
thermal hyperalgesia through EP, and EP,
receptors (78). Furthermore, it has been pro-
posed that brain-derived PGE,, at lower
doses, produces hyperalgesia through its ac-
tions on EP, receptors, whereas at higher
doses, hypoalgesia is produced through ac-
tions on EP, receptors (79-81). Further evi-
dence for involvement of EP, in mediating the
hyperalgesic effects of PGE, comes from Xin
and colleagues (82). They report that the in-
tracerebroventricular injection of GR63799X
(an EP, receptor agonist) caused fever and hy-
peralgesia in rats, which could be blocked by
an anti-sense, but not a mis-sense oligonucle-
otide directed against the EP, receptor. In ad-
dition, the use of EP knockout mice has sug-
gested that only mice lacking the EP, receptor
failed to exhibit febrile responses to PGE,, in-
terleukin-lp, and lipopolysaccharide injec-
tions, thus providing further evidence for EP,
receptor involvement in mediating the febrile
response (83).
The most recently characterized EP recep-
tor, the EP, receptor, also shows a widespread
distribution and is found in most tissues ex-
amined. It has been shown to exist in piglet
saphenous vein (29) and also in the jugular
vein of the rabbit, hamster uterus, and rat tra-
chea (84-86). In the kidney, it is expressed in
the glomerulus, mediating the effects of PGE,
on glomerular filtration. Within the brain,
EP, receptor mRNA has been found in the
hypothalamus and lower brain stem. It has
also been suggested that the EP, receptor may
be involved in bone remodeling and the induc-
tion of osteoclasts that are involved in bone
resorption. For example, Sakuma and col-
leagues (87) found that osteoclast formation
was stimulated most potently by PGE, ana-
logs that display agonist activity at EP, recep-
tors. Although it has been suggested that EP,
receptors are important in maintaining a

patent ductus arteriosus (881, EP,-'- mice
died from heart failure within 3 days of birth
of pulmonary congestion and a fully patent
ductus (89). This result remains to be ex-
plained, given the previously demonstrated di-
latatory effects in the ductus of EP, receptor
activation.
It is also interesting to note that, given the
clinical use of exogenous PGE, (dinoprostone)
in cervical ripening and labor induction (see
Section 5), the expression of the different EP
receptors has been shown to alter with time in
mice undergoing pseudopregnancy (90).
A role for E (and I) series prostaglandins in
inflammatory pain is well established, not
least because of the anti-nociceptive effects of
NSAIDS, but also because of observations that
exogenous prostaglandins can induce allo-
dynia and hyperalgesia (91). In the periphery,
sensitizing effects of PGE, have been recorded
on several aspects of sensory nerve function,
including ion channels and neuropeptide re-
lease. The EP receptors involved in mediating
these effects remain poorly characterized, but
these responses have been correlated with in-
creases in cyclic AMP, which would be sugges-
tive of EP,, EP,, or possibly, EP, receptor in-
volvement (see Section 3.4). Further studies
will hopefully address these issues and may
aid in the design of future anti-nociceptive
medicines.
2.3.3 Prostaglandin .,F Although PGF,,
can act through EP and TP receptors, it is
particularly potent at FP receptors. FP recep-
tors are mostly concentrated in corpora lutea,
where, in domestic animals, they mediate lu-
teolysis stimulated by PGF,,. It has also been
shown that, like uterine EP receptors, FP re-
ceptor expression changes during the oestrus
cycle, becoming most abundant just before the
luteal cells undergo apoptosis (92). In addi-
tion, FP receptor-deficient mice do not un-
dergo parturition even after the administra-
tion of oxytocin. Therefore, it seems that FP
receptor activation is required for normal par-
turition and labor in mice (93). This function
of PGF,, is used widely in veterinary practice,
and the receptors also seem to be present in
this capacity in humans. However, initial
hopes for FP receptor-specific agonists being
useful in controlling human fertility (94) were
Agents Acting on Prostanoid Receptors
dashed, because, unlike in animals, prosta-
glandins are not central in the destruction of
the corpus luteum. Hence, PGF,, and its ana-
logs have proved ineffective as human luteo-
lytics. However these agents are used success-
fully in animal husbandry to help synchronize
the oestrus cycles of farm animals.
Functional FP receptors have also been
demonstrated in the myometrium of some ro-
dents and also humans (95, 96). However, in
dogs, FP receptor agonists are lethal-proba-
bly as a result of the presence of contractile FP
receptors on airway smooth muscle (61). This
has yet to be reported in any other species. FP
receptors are also present in iris sphincter
muscles, and the ocular actions mediated by
these receptors have been exploited to lower
intraocular pressure and in glaucoma treat-
ment in humans.
Messenger RNA distribution studies have
identified FP receptor transcripts in the
uterus, further confirming the role of PGF,,
in this tissue. In situ hybridization has also
revealed that expression in the ovary is con-
fined to the large luteal cells of the corpus lu-
teum (97). No labeling was observed in the
ovarian follicle. Receptor mRNA was also
found in the kidney, lung, heart, and stomach,
and these studies are entirely consistent with
the actions of PGF,, in mediating luteolysis
and mesangial cell contraction in the glomer-
ulus of the kidney (98).
2.3.4 Prostaglandin I,. The primary role of
prostacyclin is thought to be in the local con-
trol of vascular tone and also the inhibition of
platelet aggregation (99). It is synthesized
mostly by vascular endothelial cells. In agree-
ment with this, IP receptors have been located
in both platelets and vascular smooth muscle
cells (100). IP receptor knockout mice, gener-
ated by homologous recombination, did not
display a hypotensive response to the potent,
selective IP receptor agonist, cicaprost (see
Section 4), but their basal blood pressure was
unaltered when compared with controls. The
mice survived normally, but following endo-
thelial damage, an increase in thrombosis was
observed, lending weight to assertions that
PGI, acts as an antithrombotic agent (67).
IP receptors have also been found on ner-
vous tissue. PGI, is a potent hyperalgesic, act-
3 Prostanoid Receptor Classification and Characterization
ing on peripheral sensory neurones and may
be more potent than PGE, in this respect, at
least in rodents (101). IP receptors have been
localized to specific subsets of dorsal root gan-
glion (DRG) neurons (102). In situ hybridiza-
tion signals were detected in 40% of DRG neu-
rons (60% small; 15-25 ~ m but ) not in glia.
Seventy percent of IP-positive neurons also
contained mRNA for preprotachykinin A
(PPTA), a substance P precursor. In addition,
around 25,41, and 24% of IP-positive neurons
co-expressed EP,, EP,, and EP, transcripts,
respectively, suggesting a possible overlap-
ping role of IP and EP receptors in mediating
pain sensation.
Mice lacking IP receptors have also been
used to suggest a role for PGI, in inflamma-
tion. For example, following carageenan ad-
ministration, IP-'- mice exhibited paw swell-
ing that was similar to that observed in wild-
type mice treated with indomethacin (67).
Similar studies have also further suggested a
role for prostacyclin in mediating peripheral
hyperalgesia (67).
2.3.5 Thromboxane A,. As far as is known,
TXA, acts exclusively through TP receptors.
TP receptors exhibit a widespread distribu-
,tion in platelets and vascular smooth muscle,
where they mediate platelet aggregation and
'also smooth muscle contraction (103,104). TP
lreceptors are also present in airway smooth
!muscle (105), where they mediate broncho-
constriction. Several studies have examined
the distribution of TP receptor mRNA. In the
mouse, TP receptor transcripts have been de-
d abundantly in the thymus, spleen, and
g (106). In human tissues, TP receptor
anscripts were found in the placenta and the
ng (107). Therefore, it seems that TP recep-
rs are present in immune related organs
e.g., spleen and thymus) as well as those rich
smooth muscle, such as the lung. Ushikubi
d colleagues (108) have further investigated
TP receptor population in the thymus. Us-
radioligand binding, they found TP recep-
expression was greatest in immature
4-8- and CD4+8+ cells, and that recep-
expression decreased as cells matured.
also suggested the presence of functional
receptors on immature thymocytes, as the
275
addition of a TP receptor agonist induced ap-
optosis in this cell population that was sensi-
tive to TP receptor antagonism. Hence, in ad-
dition to roles in the cardiovascular and
respiratory systems, TP receptors may also
act in thymocyte development.
Recently, mice deficient in TP receptors
have been generated to further elucidate po-
tential roles of thromboxane A,. These TP-'-
mice exhibited an increased tendency to bleed
and were unresponsive to the intravenous in-
jection of the TP receptor specific agonist,
U46619 (109). The increase in bleeding ten-
dency has also been reported in human pa-
tients with a point mutation (Arg-60 to Leu) in
the first cytoplasmic loop of their TP receptor
(110). These patients display a defective plate-
let aggregatory response to TXA,, which fur-
ther suggests a role for TP receptors in hemo-
stasis.
3 PROSTANOID RECEPTOR
CLASSIFICATION AND
CHARACTERIZATION
The general scheme proposed by Kennedy and
colleagues (see Section 1) is still in use today
(11,and has been confirmed and expanded by
the use of modern techniques in molecular bi-
ology, which have made possible the cloning
and molecular characterization of all the
known prostanoid receptors.
The current system terms prostanoid re-
ceptors P receptors, which is preceded by a
letter to indicate the most potent natural pro-
stanoid at that particular receptor (hence the
receptor at which PGD, is the most potent
natural agonist is called the DP receptor and
so on). This system was based initially upon a
rigorous quantitative comparison of the ago-
nist potencies of each of the natural prosta-
noids in tissue preparations chosen to avoid
the problems associated with multiple recep-
tor types. In addition, being aware of the lim-
itations of agonists in receptor classification
( I l l ) , efforts were made to identify specific
prostanoid-receptor antagonists. A summary
of the current state of prostanoid receptor
classification is shown in Table 6.2.
276 Agents Acting on Prostanoid Receptors

Table 6.2 Second Messenger Couplings, Amino Acid (AA) Numbers, and Chromosomal
Locations of the Cloned Prostanoid Receptors
hceptor Number Chromosomal
Receptor Subtype Isoforms Coupling Species of AAa Location Refs.
Human 359 N.D.
G,. T cAMP Mouse 357 14
Rat 357 N.D.
Human 402 19~3.1
? [Ca2+li Mouse 405 8
through
unidentified
G-protein
Rat 405 N.D.
Human 358 5p13.1
G,, T CAMP Mouse 362 N.D.
Rat 357 N.D.
Human 365 lp31.2
Gi, .1 Mouse 365 3
CAMP^
Rat 366 N.D.
Human 488 5p13.1
G,, T 'AIvlP Mouse 513 15
Rat N.D.
Human 358 lp31.1
G, T Mouse 366 3
[Ca2+li
with T PI
turnover
Rat 366 N.D.
Human 386 19q13.3
GJG, and T Mouse 417 7
CAMP/T
IP3
Rat 416 N.D.
Human 343 19~13.3
G,, T Mouse 341 10
[Ca2+li
with PI
turnover
Rat 341 N.D.
"Owing to C-terminal splicing, amino acid numbers are variable for EP, receptors. The AA numbers shown are for the
hEP3.,II receptor, the mEP,, receptor, and rEP,, receptors.
bMultipleC-terminal splice variants of EP, receptors exist that couple to different G-proteins. For example, bovine EP,
receptors can couple to Gi, but also to G,and G,.
PI, phosphatidyl inositol; f , increase; 4,decrease.

3.1 Prostanoid Receptor Subtypes equivocally. However, recently, it has been

and lsoforms
Although the subclassification of EP receptors
is well accepted and verified by numerous
studies, the evidence for subtypes of DP, IP,
and TP receptors is less clear. There is some
evidence for two subtypes of DP receptor (129,
130), although this has yet to be proven un-
shown that PGD, may act through a novel,
putative chemoattractant receptor, termed
CRTH2, which is preferentially expressed in
human T helper type 2 cells, eosinophils, and
basophils (131). This receptor is particularly
interesting in that it is not a member of the
general prostanoid receptor family; rather, it
 3 Prostanoid Receptor Classification and Characterization
is related to other chemoattractant receptors,
such as that for N-formylmethionyl-leucyl-
phenylalanine (fMLP). Although PGD, clearly
has affinity for this receptor, it is not clear
whether it is the natural ligand, and so for the
time being, its identification as a bone fide
prostanoid receptor must remain tentative.
There is also some functional evidence for a
subdivision within IP receptors (19, 132). For
example, Wise and colleagues (133) found the
PGI, mimetic, BMY45778, to be a potent and
full IP receptor agonist in human platelets and
rat neutrophils. In contrast, this compound
was a weak partial agonist in the rat colon,
suggesting the possible existence of more than
one subtype of IP receptor.
Although it is unclear whether there are
several subtypes of TP receptors (134, 135),
this receptor does seem to have at least two
isoforms, generated by alternative splicing,
both of which have been cloned in humans
(136).These differ in the lengths of their C-
terminal tails distal to Arg-328. Similar alter-
native splicing has been reported in both EP,
(I),EP, (1371, and FP receptors (138). This
been particularly well characterized in the
e of the EP, receptor. The EP, receptor
sts in several splice-variants, and it has
een proposed that at least six splice-variants
uld exist in each species. These variants all
cur at the same relative amino acid in the
-terminus of the EP, receptor, thus altering
e length of the C-terminal tail. This has re-
ntly been shown to alter potential signal
sduction pathways and localization of the
receptor (118, 139, 140). Despite these
rences, the binding affinities for the nat-
al prostanoids seem similar, and it is un-
ear at present whether other EP receptor
lective ligands display differences in affinity
d agonist potency between different EP, re-
I
ceptor splice-variants, because no detailed
comparative studies have been carried out un-
der similar experimental conditions.
I
' 3.2 Prostanoid Receptors as
C-Protein-Coupled Receptors
The first prostanoid receptor to be cloned was
the TXA, receptor (107), and since then, ho-
mology screening has been used to clone pro-
stanoid receptors from several species, includ-
ing mice, rats, humans, and cows (137).
277
Hydrophobicity and homology analyses of
these receptors have revealed them to have
seven putative transmembrane domains, sug-
gesting they are all G-protein-coupled, rho-
dopsin-like receptors (GPCRs) belonging to
the main subclass (2.1) (141). Overall homol-
ogy between the different receptor types is
quite low (20-30%), whereas homology be-
tween species orthologues is much higher (70-
90%) (137). A summary of the gene locations
and amino acid lengths of the cloned prosta-
noid receptors is shown in Table 6.2. Recent
reports have also suggested that functional
prostanoid receptors are present at the nu-
clear envelope, as well as at the cell surface.
For example, nuclear EP, receptors can in-
duce increases in intranuclear Ca2+ and in-
ducible nitric oxide synthase (iNOS) gene ex-
pression (142, 143).
Despite the structural differences in recep-
tor subtypes, strong conservation exists in
several areas. There are 28 residues conserved
around the putative transmembrane domains,
8 of which are shared by other families of
GPCRs, and these are believed to be involved
in structure/function relationships of GPCRs
in general. Thus, two cysteine residues in the
first and second intracellular loops are con-
&
served, and these may form a disulphide
bridge necessary for the stabilization of recep-
tor conformation to allow ligand binding
(144). Also conserved is an aspartate. in the
A
second transmembrane region, which has
been shown in other receptors to be important
in coupling ligand binding to G-protein activa-
tion (145). Concensus sequences for N-glyco-
sylation are also present, for example, in the
second extracellular loop of EP, and EP, re-
ceptors. In addition, serine and threonine res-
idues, putative phosphorylation sites, are
widely distributed in the cytoplasmic portions
of the receptors and may play a role in receptor
desensitization, as has been reported for hu-
man TP receptors (146,147).
Specific to the prostanoid receptors, the
most conserved region is the seventh trans-
membrane domain, where the sequence L-X-
A-X-R-X-A-SIT-X-N-Q-I-L-D-P-W-V-Y-I-L is
shared. Two further sequences, G-R-Y-X-X-Q-
X-P-G-T/S-W-C-Fand M-X-F-F-G-L-X-X-L-L-
X-X-X-A-M-A-X-E-R, are also present in the
second extracellular loop and third transmem-
278
brane domain, respectively. These have been
suggested to be involved in prostanoid bind-
ing, with particular attention being paid to the
conserved Arg in the seventh transmembrane
domain, which is located at the analogous po-
sition to the retinal binding site, LysZs6,of the
rhodopsin receptor. It was suggested that this
residue might serve as the binding site for the
o-carboxyl group of prostanoid molecules
(148). Site-directed mutagenesis experiments
have also been conducted to elucidate the
binding sites for various prostaglandin recep-
tor-selective ligands, and these aspects will be
covered in more detail (Section 4).
Computer-assisted sequence analyses have
been used to construct phylogenetic trees to
infer the evolutionary pathways of the prosta-
noid receptors. For example, Toh and col-
leagues (149) examined the molecular evolu-
tion of the lipid mediator receptors. They
reported prostanoid receptors forming a dis-
tinct cluster within the rhodopsin-like
GPCRs. In contrast, the receptors for platelet
activating factor and lipoxygenases were in
another cluster with the receptors for tachyki-
nins and bradykinin. Moreover, the prosta-
noid receptor cluster could be further subdi-
vided (Fig. 6.3). Hence, the DP/EP,/EP, and
IP receptors, which predominantly exert their
effects through increasing intracellular CAMP
levels, seem to be more closely related to each
other than to EP,/FP and TP receptors, which
predominantly act through increasing intra-
cellular Ca2+,or to EP, receptors, which, al-
though often causing decreases in intracellu-
lar CAMP,have been reported to act through
multiple signaling pathways (see Section
3.4.4). Other groups (117, 149) have also pro-
posed similar evolutionary pathways. These
studies are consistent with the proposal that
the PGE system and receptor may have
evolved before the individual EP receptor sub-
types, which mediate specific signaling path-
ways. Additionally, the construction of phylo-
genetic trees suggests that the prostanoid
receptors evolved separately from the recep-
tors for other lipid-signaling molecules.
3.3 Radioligand Binding Studies
The proposed scheme of receptor classification
has been further supported by the existence of
high affinity binding sites for each natural
Agents Acting on Prostanoid Receptors
prostanoid, both in native tissues and heterol-
ogous systems. These studies have also been
furthered by the generation of radioligands
from ligands that show selectivity for particu-
lar prostanoid receptors, such as the recent
development of [3Hl-BWA868C to detect DP
receptor-like binding sites (150).
3.3.1 PGD2-Specific Binding Sites. [,HI-
PGD, binding sites have been identified in hu-
man platelets, and these sites are bound, with
high affinity, by the DP receptor selective ag-
onist, BW245C (see Section 12). In contrast,
BW245C does not displace [3Hl-prostacyclin
binding from human platelets, suggesting that
the binding sites are different. Likewise, the
other natural prostaglandins show lower af-
finities in displacing r3H]-PGD, binding than
PGD, or BW245C (151,152). Thus, these data
are consistent with functional studies suggest-
ing that platelets, in humans at least, contain
DP receptors. Binding sites for [,HI-PGD,
have also been reported in synaptic mem-
branes from the rat (153). Recently, [,HI-
BWA868C has been reported and character-
ized as a new radioligand to aid the
identification of PGD, binding sites (150).
This radioligand showed high affinity binding
in human platelets that were displaced by
other ligands known to act through the DP
receptor. Under the same conditions, EP, FP,
IP, and TP receptor selective ligands were
poor competitors, with Ki values in the micro-
molar range. This could be an important ad-
vance, because the use of a specific DP recep-
tor antagonist should allow the closer
correlation of affinities and potencies from
functional studies, without the added compli-
cations of differing agonist efficacies between
preparations.
3.3.2 PGE2-Specific Binding Sites. The use
of [,HI-PGE, as a radioligand has proved useful
in the determination of PGE, binding sites that
may or may not represent functional EP recep-
tors. However, because of the non-selective na-
ture of this ligand and also the lack of truly se-
lective EP receptor agonists and antagonists,
this approach to subclassifyingEP receptor sub-
types has been limited. Nevertheless, high affin-
ity binding sites (pK, > 8) have been reported in
adipocytes, brain, dorsal root ganglia, kidney,
3 Prostanoid Receptor Classification and Characterization 279

Swiss-prot
accession
no.

q62928
q62053
p43116
q9xt82
q9r261
035932
~70263

Figure 6.3. Molecular grouping of the prostanoid receptors. The dendogram was constructed from
sequence comparison of the published sequences of the cloned prostanoid receptors from various
species. The sequences used are identified by their accession numbers shown within the figure. The
length of the solid lines represent evolutionary diversity.

corpus luteum, myometrium, gastrointestinal
tract, liver, heart, and platelets (32). Further-
more, in some cases, PGE analogs have been
used to attempt to identlfy the EP subtype
involved. For example, competition studies us-
ing [3H]-PGE, and sulprostone or AY23626
conducted in membranes prepared from rat adi-
pocytes and renal collecting tubules have sug-
gested the presence of EP, receptors. Encourag-
ingly, the binding a n i t i e s obtained under
these conditions can be correlated to the relative
agonist potencies obtained for these compounds
in other prototypical EP,, EP,, and EP, recep-
tor-containing tissues.

3.3.3 PGF2,-Specific Binding Sites. The
majority of studies using [3H]-PGF2, have
been conduced on the corpora lutea of several
species, including the rat (1541, rabbit (1551,
and sheep (156). The detection of high affinity
binding sites in these areas is entirely consis-
tent with the effects of PGF,, as a luteolytic
agent in several species of animal and suggests
the presence of functional FP receptors in me-
diating prostaglandin-induced luteolysis. In
addition, this binding is displaced specifically
by unlabeled PGF,,, and studies using flupro-
stenol and cloprostenol, both highly selective
and potent PGF analogs (see Section 4.31, re-
veal a good correlation between binding affin-
ity and agonist potency at FP receptors. In
addition, when expressed in recombinant sys-
tems, there seems to be a good correlation be-
tween rank orders of affinity between recom-
binant FP receptors and those believed to be
present in corpora lutea (157). [3Hl-17-phenyl
PGF,, has also been reported as a radioligand
useful in the determination of FP receptor
binding sites (158).
3.3.4 PG12-Specific Binding Sites. IP recep-
tors have been identified in a wide variety of
tissues, and PGI binding sites reflect this.
Hence, binding studies have been performed
on platelets, vascular smooth muscle, and
nerve cells, as well as the guinea pig lung, gen-
erally using [3H]-prostacyclin, [3H]-iloprost,
or [3H]-PGEl as the radioligand (159, 160).
In all the preparations studied, a similar
order of agonist potency was obtained in com-
petition studies. Hence, iloprost = PGI, >
PGE, (10-fold weaker) > PGD, = PGE, =
PGF,, (32). Furthermore, if PGD,, for which
there are known binding sites on human blood
platelets (161), is omitted, there is a clear as-
sociation between the rank order of competi-
tive potency and the relative potencies of these
agonists to inhibit platelet aggregation. These
data thus suggest that the [3H]-PG12binding
sites identified in the above tissues are likely
to be IP receptors of the same type.
In recombinant systems, the rank orders of
competitive potency for a wide range of pros-
taglandin receptor ligands are similar to those
obtained in platelets and other tissues, thus
providing further evidence for the existence of
IP receptors in these tissues (126, 162-164).
Agents Acting on Prostanoid Receptors
3.3.5 TXA2-Specific Binding Sites. Radioli-
gand binding studies have been performed us-
ing both TP receptor selective agonists and
antagonists. Agonists used include r3H1-
U44069 (165), r3H]-U46619 (166), and [12511-
I-BOP (167), and antagonists used include
[3H]-13-APA, [1251]-PTA-OH, and more re-
cently, [3H]-SQ29548 (1681, [3Hl-GR32191B
(169),and [1251]-S-145(170),all of which have
pK, values of >8. The latter compounds are
particularly potent TP receptor blockers and
are thus ideally suited for use in radioligand
binding studies. Several studies have been
performed on platelets, and in general, a good
correlation in ligand potencies has been ob-
served between functional and binding stud-
ies. For example, in human platelets, L3H1-
U44069 binding was displaced by U46619 and
also TXA, (171). Similarly, identical rank or-
ders have been obtained for the TP receptor
antagonists, SQ29548, 0N03708, BM13177,
and 13-APA in human platelets using both
functional and ligand binding techniques
(166). Interestingly, the binding properties
of r3H]-SQ29548 and [3Hl-GR32191B (va-
piprost) seem to reflect some of their pharma-
cological properties in man. Thus, [3Hl-SQ29548
shows rapid association and dissociation ki-
netics (168),whereas [3H]-GR32191Bexhibits
slow dissociation kinetics (169), which corre-
lates with this compound's pharmacological
profile in platelets where it seems to act as a
partially insurmountable receptor blocker
(172). The binding properties of recombinant
TP receptors have also been extensively exam-
ined and the rank orders of affinity obtained
are in good agreement with those obtained in
membranes derived from endogenous TP re-
ceptor-containing tissues (157,164).
3.3.6 Summary. Thus, it can be seen that
specific binding sites for all of the main natu-
ral prostaglandins have been identified in both
membranes prepared from tissues and also in
recombinant systems. These studies, showing
binding sites at which each of the natural pro-
stanoids displays the highest binding affinity,
are consistent with the proposed system of re-
ceptor classification. Likewise, the good corre-
lation between the orders of affinity in compe-
tition studies and agonist potency from
functional studies suggest that binding sites
3 Prostanoid Receptor Classification and Characterization
identified are functional receptors and that ra-
dioligand binding is a useful tool to aid in re-
ceptor identification and classification.
3.4 Signal Transduction
The coupling of prostanoid receptors to intracel-
lular signalling pathways has been examined by
several methods using both recombinant sys-
tems and in vitro preparations to determine the
Gproteins and second messengers involved.
3.4.1 D P Receptors. DP receptors couple
through G, to increases in intracellular cyclic
AMP. Indirect evidence for this was obtained
by Simon and colleagues (173), who showed
that D, E, and I series prostaglandins all stim-
ulated adenylate cyclase in human colonic mu-
cosa. These early observations have been con-
firmed by studies using recombinant DP
receptors, where it has been demonstrated
that both PGD, and the DP receptor selective
agonist, BW245C, concentration dependently
increase cyclic AMP levels in Chinese hamster
ovary (CHO)cells expressing recombinant DP
eceptors (49, 112).
3.4.2 EP, Receptors. The G-protein in-
lved in EP, receptor signaling has yet to be
entified (1). Studies in recombinant cells
ggest the involvement of Ca2+ mobilization.
atabe and colleagues (64) showed PGE,
sed an increase in intracellular [Ca2+]in
HO cells expressing this receptor, an event
at was dependent on extracellular Ca2+ and
ompanied by a very weak inositol trisphos-
ate (IP,) response. This agrees with Creese
d Denborough (174) who showed a similar
racellular Ca2+ effect in guinea pig tra-
hea, a preparation known to contract
hrough EP, receptors (175).
3.4.3 EP, Receptors. EP, receptors are
upled to G, and mediate increases in intra-
llular CAMP concentrations. Evidence for
his was first obtained by Hardcastle and co-
rkers, who reported a positive association
ween EP, and CAMP generation in entero-
es (176) and more recently from recombi-
3.4.4 EP, Receptors. EP, receptors have
n shown to couple to several different sig-
281
nal transduction systems. The major coupling
is to G, to reduce adenylate cyclase activity,
but there are several reports of differential
coupling of splice variants, including coupling
to increases in intracellular calcium (139).
This is consistent with responses observed in
tissues, where EP, receptor activation can in-
hibit gastric acid secretion (see below) and li-
polysis, two actions classically mediated by de-
creases in cellular CAMP. In addition, it also
seems that EP, receptors can mediate smooth
muscle contraction, consistent with increases
in intracellular Ca2+(27). In addition, the EP,
receptor seems to be involved in the inhibition
of arginine vasopressin water reabsorption
through a pertussis toxin-sensitive pathway,
suggesting the involvement of Gi (177, 178).
Likewise, the EP,-mediated inhibition of acid
secretion in the stomach is also pertussis-sen-
sitive (72).
Four EP, receptor isoforms have been
cloned from cDNA generated from a bovine
adrenal gland, and these display coupling to
multiple second messenger pathways. Thus,
the bovine EP3, isoform couples to G,, the
EP,, and EP,, isoforms to G,, and the EP3,
isoform to G,, G,, and G, (179). Isoforms of the
mouse EP, receptor, EP,, and EPSp,differ in
their responses to GTPyS and also in potency
at inhibiting forskolin-induced CAMP accu-
mulation, with EP3y requiring threefold
lower concentrations of agonist than EP,@to
evoke a 50% inhibition (119).
3.4.5 EP, Receptors. This receptor sub-
type, like the EP, receptor, is known to couple
to G, and mediate increases in intracellular
CAMP levels (116). There is some confusion in
the early literature regarding the identity of
the "true" EP, receptor. The "EP," subtype,
originally cloned by Honda and colleagues
(122) and Bastien and co-workers (121) has
since been identified as the EP, receptor, on
the basis of its insensitivity to the EP, recep-
tor-selective agonist, butaprost (see Section
4), and sensitivity to the EP, receptor antago-
nist, AH23848B. The EP, receptor cloned by
Regan and colleagues (117) is regarded as the
"true" EP, receptor subtype.
3.4.6 FP Receptors. This receptor type me-
diates increases in inositol triphosphate for-

mation through G, and phospholipase C acti-
vation. It has been known for some time that
stimulation with PGF,, is coupled to in-
creases in phosphoinositide turnover and the
elevation of intracellular calcium (180, 181).
In mouse fibroblasts, the effect of PGF,, to
elevate intracellular calcium is pertussis toxin-
insensitive, suggesting a lack of involvement
of G,, and correlates with IP, formation (182).
Similar results have been obtained using re-
combinant FP receptors, where FP receptor
activation resulted in a concentration-depen-
dent increase in IP, formation (97).
3.4.7 IP Receptors. IP receptors mediate
an increase in CAMP levels through G, (163),
but recombinant studies in CHO cells have
also revealed a phosphatidyl inositide re-
sponse that is cholera- and pertussis toxin-
insensitive, suggesting the involvement of G,
(126).
3.4.8 TP Receptors. TP receptors are gen-
erally regarded as signaling through G, to
cause an increase in the concentration of in-
tracellular calcium (183). However, there are
reports that TP receptors may also couple to
GI,, GI,, and G13 (184-186). Furthermore,
there have also been reports that TP receptor
splice variants, while both coupling to G,, may
interact differently with adenylate cyclase.
Thus, the TPa receptor isoform induces in-
creases in intracellular CAMP, whereas the
TPP isoform induces decreases in intracellu-
lar CAMPlevels (187).
4 PROSTANOID RECEPTOR-SELECTIVE
LICANDS AND STRUCTURE-ACTIVITY
RELATIONSHIPS
From the desire to produce novel therapeu-
tics, several ligands that are selective for sub-
types of prostaglandin receptors have been de-
veloped, and these are detailed below.
4.1 DP Receptors and Selective Ligands
The structures of some DP receptor ligands
are shown in Fig. 6.4. PGD, is not an inher-
ently selective agonist; it also has FP and TP
receptor activity (188). The dehydration prod-
uct of PGD,, PGJ, (9-deoxy-A9-PGD,), (189)
Agents Acting on Prostanoid Receptors
is equipotent to the natural agonist in produc-
ing vasodilation and inhibiting of platelet ag-
gregation and more selective with respect to
EP and FP receptor activity. PGJ, has also
been shown to be of similar potency to PGD,
in heterologous systems (190). Furthermore,
PGJ, and its derivative, 15-deoxy,12,14-PGJ,,
are also agonists at the peroxisome prolifera-
tor-activated receptor-gamma (PPARy) (191).
The most widely described DP receptor ago-
nist is the synthetic prostanoid, BW245C
(192). This is a hydantoin prostanoid analog
that is at least one order of magnitude more
potent than PGD, as a DP receptor agonist,
but is considerably less active at TP and FP
receptors. It displaces [,HI-PGD,, but not
[3Hl-prostacyclin binding form in bovine
platelets (1521,and its inhibitory action on hu-
man platelets can be blocked by the DP recep-
tor antagonist, AH6809. Furthermore, BW245C
has been shown to potently inhibit the aggre-
gation of human platelets and relax the rabbit
transverse stomach strip (193). Another DP
receptor selective agonist is the 9-chloro ana-
log of PGE,, ZK110841 (194). SQ27986 has
also been described as a potent and highly se-
lective DP receptor agonist (195). Interest-
ingly, introducing a 15-cyclohexyl group into
the a-side chain of PGD, and other prosta-
glandins seems to enhance DP receptor-like
selectivity. Finally, and most surprisingly, is
RS93520. This is an analog of PGI,, but is far
more potent as a DP receptor agonist, being
only a modest IP receptor agonist on the hu-
man platelet (196). Recently, L-644698 has
been described as a novel DP receptor selec-
tive agonist (190), and it has been reported to
be at least 300-fold more selective for human
DP receptors over human EP, receptors and
more than 4000-fold more selective over the
other human recombinant prostanoid recep-
tors. It also exhibits similar efficacy to PGD,
and BW245C, as evaluated by the accumula-
tion of CAMP in recombinant cells, suggesting
this may prove a useful tool in DP receptor
characterization in the future. The agonist
and antagonist potencies of some ligands ac-
tive at the DP receptor are shown in Table 6.3
and affinity constants at both mouse and hu-
man recombinant receptors in Table 6.4.
The first compounds with recognized DP
receptor blocking activity were the phloretin
 Receptor-Selective Ligands and Structure-Activity Relationships

Agonists
HO
5

Antagonists
0

Figure 6.4. The structures of some DP receptor-selective ligands.

284 Agents Acting on Prostanoid Receptors

Table 6.3 The Potencies of Some DP Receptor Agonists and Antagonists

Equieffedive Concentration
Agonists Relative to PGD2 (=1.0) Refs.

p~ - -

Antagonists PA, Refs.

derivatives, N-0164, N-0057, and N-0161.
N-0164 antagonized the anti-aggregatory ac-
tions of PGD, and BW245C on human plate-
lets and also inhibited the concomitant PGD,-
induced CAMP elevation (201). However,
N-0164 also possesses activity in other sys-
tems. For example, it antagonizes PGE,- and
PGF,,-induced gastrointestinal smooth mus-
cle contraction (202) and has also been shown
to have inhibitory effects on thromboxane
synthase (203) and CAMP phosphodiesterases
(2041, in addition to blocking TP receptors

Table 6.4 Affinities of Some Prostanoid Receptor Agonists and Antagonists (*) at
Recombinant Human and Mouse DP, FP, IP, and TP Receptors
Human [K, (nM)] Mouse [Ki (nM)I

Ligand DP FP IP TP DP FP IP TP
PGE, 307 119 ** >10000 *** *** *** ***
SC19220* N.D. N.D. N.D. N.D. *** *** *** ***
Butaprost FA >10000 ** >10000 >10000 *** *** *** ***
Butaprost ME >10000 >10000 >10000 >10000 N.D. N.D. N.D. N.D.
Sulprostone ** 198 ** ** *** 580 *** ***
M&B28767 \glOOOO 510 \glOOOO 343 *** 124 *** 1300
GR63799 >10000 1241 >10000 >10000 *** *** *** ***
Enprostil >10000 88 >10000 >10000 *** *** *** ***
Misoprostol FA >10000 >10000 ** ** *** *** *** ***
Misoprostol ME ** ** ** ** N.D. N.D. N.D. N.D.
11-deoxy-PGE, N.D. N.D. N.D. N.D. +** *** 1000 ***
AH23848B* 1380 >10000 >10000 592 N.D. N.D. N.D. N.D.
PGD, 1.7 6.7 ** 6602 21 47 *** ***
BW245C 0.4 >10000 >10000 >10000 250 1700 *** ***
ZK110841 0.3 1670 2138 1121 *** *** *** ***
BWA868C* N.D. N.D. N.D. N.D. 220 *** *** ***
PGF2, 861 3.2 >10000 8700 *** 3.4 *** ***
Fluprostenol ** 2.3 ** >10000 *** 3.8 *** ***
Cloprostenol >10000 0.47 ** 6123 N.D. N.D. N.D. N.D.
Latanoprost 555 ** ** >10000 N.D. N.D. N.D. N.D.
Iloprost 1035 619 11 6487 *** *** 11 ***
Cicaprost >1340 >I340 17 >I340 *** *** 10 ***
Carbaprostacyclin 132 360 282 >10000 *** *** 110 ***
U46619 3970 241 >10000 35 1000 *** *** 67
SQ29548* *L ** ** 4.1 *** *** *** 13
Human data taken from Abramovitz et al. (157).Mouse data from Kiryama et al. (164).
**Ki estimate > 100,000 nM.
***Unablet o displace 50% of their radioligand at 10 p M .
4 Prostanoid Receptor-Selective Ligands and Structure-Activity Relationships
(205).Indeed, N-0164 was first identified as an
EP receptor blocker, where it antagonizes
PGE, and PGF,,-induced smooth muscle con-
tractions (200). AH6809 is also a DP receptor
blocker, but this compound also exhibits an-
tagonist activity at TP, EP, (and human EP,)
receptors (164,206), having an estimated PA,
value of 6.5 in both cases. A more potent and
selective DP receptor antagonist was de-
scribed by Giles and colleagues (200):
B W A ~ ~ ~aCderivative
-is of the D P receptor
agonist, BW245C and is the most useful DP
receptor antagonist currently available. It has
also been shown to possess activity in vivo
(207). This compound displays potent antago-
nist activity against the functional effects of
PGD, in human platelets (2081, rabbit jugular
and saphenous vein (200, 2091, and human
myometrium (210). Recently, S-5751 has also
been described as a DP receptor selective an-
[3H]-BWA868Chas also recently been de-
scribed (195). This radioligand displayed high
aMinity binding in human platelets and the DP
ive ligands described above all
affinities in competition exper-
ame tissue. Ligands active at
anoid receptors were, in con-
petitors. Thus, it seems that
ay prove useful for future DP
erization and autoradio-
.2 EP Receptors and Selective Ligands
4.2.1 General Considerations. The identifi-
ion of PGE analogs with selective func-
trates the progress that has
n made into determining structure-activ-
relationships for EP receptor selectivity,
it seems that further structural features
fer EP receptor subtype selectivity. For EP
nist activity, the length of the a-chain is
teration of this by a single
ring loss of EP receptor se-
ty (212). Substitutions at C-1 seem to
uce agonist potency at EP, receptors.
e carboxylic acid group
for example, esters, methanesulfon-
ups, seems to maintain
and EP, receptors only
t, substitutions at C-2 to
C-6 considerably reduce agonist potency at EP
receptors. Furthermore, an alkynic link be-
tween C4 and C5 reduces EP, agonist potency,
whereas an interphenylene at C-3-C-7 in-
creases selectivity for the EP, receptor (215,
216). The cyclopentyl ring also seems critical
for EP receptor selectivity, because substitu-
tion with a cyclohexyl group produces a signif-
icant loss of activity at EP receptors in general
(212). Likewise, a carbonyl group at C-9 is pre-
ferred for EP receptor agonist activity, al-
though a methylene group allows EP, and EP,
receptor activity (217-219). The hydroxy
group at C-11 seems critical for EP, receptor
activity, but not at other EP receptors. How-
ever, removal of this group seems to decrease
agonist potency at all EP receptor subtypes
and may also result in a relative increase in TP
receptor agonist activity.
The length of the o-chain does not seem
crucial in conferring EP receptor agonist ac-
tivity, and there are several cases in which the
length of this chain has been altered without
inducing differences in agonist activity (32).
Likewise, the C-13-C-14 double bond is not
critical, as long as it is present in the trans
conformation. In contrast, a hydroxy group at
C-15 is necessary for EP receptor agonist ac-
tivity, but the presence of this group at both
C-15 and C-16 can reduce EP, receptor po-
tency. When a methyl group is introduced at
C-15 or (2-16, TP receptor activity seems to
increase (220). The substitution of the final
two to four carbon atoms of the methyl chain
with a cyclic structure also seems to confer
some EP receptor selectivity, with 17-phenyl-
trinor-PGE, probably one of the best-docu-
mented examples. This agonist has some EP,
receptor selectivity (32).
Recently, Ungrin and colleagues (221) per-
formed a study to determine the structure ac-
tivity relationships of 55 prostanoid-like com-
pounds at the recombinant human EP,
receptor. They examined the effects of these
analogs on both receptor binding and activa-
tion. On the basis of this, they suggested that
the C-11 and C-15 hydroxy group are essential
for agonist activity, along with the presence of
a carbonyl group at C-9, rather than an ester
moiety. Furthermore, they found that the al-
teration of the w-tail could enhance the po-
tency of otherwise weak compounds. In sum-

1 7-phenyl-trinor-PGE2
Antagonists
0
II
AH6809
Figure 6.5. The structures of some EP, receptor-selective ligands.
m a y , in conferring EP receptor selectivity,
the following elements are important: an
a-chain of seven carbon atoms, a cyclopentyl
ring substituted at C-9, preferably with a car-
bony1 group, and a hydroxy group at C-15 or
C-16.
4.2.2 EP, Receptor-Selective Ligands. The
structures of some EP, receptor selective li-
gands are depicted in Fig. 6.5. To date, no
highly selective, potent EP, receptor agonists
have been described, and it is only recently
that potent EP, receptor antagonists have
been reported. 17-phenyltrinor-PGE (see
above), 16,16-dimethyl PGE,, ICI 80205, and
9-methylene PGE, all show moderate EP, re-
Agents Acting on Prostanoid Receptors
sulprostone
SC- lW2O
ceptor selectivity but are also active at other
EP receptors (222). ICI 80205 also has TP re-
ceptor activity. Sulprostone was also identi-
fied as an EP, receptor selective agonist, but it
is actually more potent as an EP, receptor se-
lective ligand (157). Iloprost is a stable analog
of PGI,, but this behaves as a potent partial
agonist at EP, receptors. Thus, compounds do
not have to be PGE derivatives to display EP,
agonist activity. Iloprost is also relatively se-
lective for EP, receptors over the other EP
receptor subtypes, displaying very little func-
tional activity in EP, and EP, containing
preparations.
Several compounds have been synthesized
that demonstrate EP, receptor blocking activ-
4 Prostanoid Receptor-Selective Ligands and Structure-Activity Relationships
ity in functional studies. The first of these
compounds to be described is the weak antag-
onist SC-19220 (223), with PA, values of 5.2-
5.6 in EP, receptor-containing preparations.
This compound and its derivatives have been
tested for EP, antagonist activity in both the
guinea pig ileum and the rat fundus. It was
found that increasing the length of the acetyl
chain increases potency but reduces selectiv-
ity. SC-19220 has also been found to be active
in vivo, showing antinociceptive effects in a
variety of animal pain tests (224). However,
SC-19220 is highly insoluble and has also been
shown to exhibit some local anesthetic activity
(225). Although SC-19220 is regarded as a
weak competitive EP, antagonist on the basis
of functional studies, it has negligible binding
affmity for the recombinant mouse EP, recep-
tor (164).AH6809 also shows weak EP, block-
ing activity, although it is more potent than
SC-19220. However, this compound is also a
blocker of the human EP, receptor (206) and
DP receptors. No binding of this compound to
the recombinant mouse EP, receptor was de-
teded, and at the human receptor, AH6809,
displayed similar weak affinities for EP,, EP,,
EP,, and DP receptors (157,164). In addition,
the usefulness of AH6809, especially in vivo, is
compromised by its avid binding to albumin; it
is approximately 97% protein bound in the
presence of 4% bovine serum albumin (226).
Recently, Shaw and colleagues have reported
anew EP, antagonist, ZM325802, which they
have shown to have a K,of 0.25 nM at human
recombinant EP, receptors and to be 8400-
fold more selective over EP, and EP, recep-
tors. Binding at EP, receptors was not deter-
Table 6.5 Potencies of Some EP, Receptor Agonists and Antagonists
Equieffective Concentration
Relative to PGE, (=LO)
17-phenyl-trinor PGE,
287
mined, but this compound failed to antagonize
the relaxant effect of PGE, in the guinea pig
isolated-trachea preparation, a model of EP,
receptor activity (63). They also found that
this compound displayed anti-nociceptive
properties in in vivo tests in the rat, further
suggesting a possible role for EP, receptors in
prostaglandin-induced hyperalgesia. A sum-
mary of the agonist and antagonist potencies,
as well as the inhibition constants of ligands
that have EP, receptor activity, are shown in
Tables 6.5 and 6.6, respectively.
It should be noted that, whereas most of
the agonists described at EP, receptors are
obvious derivatives of PGE, (with the excep-
tion of iloprost), none of the antagonists thus
far described bear any clear chemical resem-
blance to E series prostaglandins.
4.2.3 EP, Receptor-Selective Ligands. The
structures of some compounds that are active
at EP, receptors are shown in Fig. 6.6. Several
compounds have been described that seem to
be specific EP, receptor agonists. The first
compound identified, but later found to pos-
sess some EP, receptor activity as well, was
the PGE, analog, AY23626 (228). Misoprostol
and 19(R)-OHPGE, (229) are also EP, recep-
tor agonists, but the former is more potent at
EP, receptors (222) and the latter at EP, re-
ceptors (116). Despite a lack of absolute selec-
tivity, both these agents have been useful tools
in preparations lacking EP, receptors. More
specific for the EP, receptor are AH13205
(230) and butaprost (TR4979) (231). Butap-
rost, although not possessing great potency, is
highly selective for the EP, receptor subtype
Refs.
1 227
4-6 228
la 60,116
P& Refs.
5.2-5.6 226,228
6.67.0 226,228
288 Agents Acting on Prostanoid Receptors

Table 6.6 Affinities of Some Prostanoid Receptor Agonists and Antagonists (*) at
Recombinant Human and Mouse EP,, EP,, EP,, and EP, Receptors
Human [K,
(nM)] Mouse [Ki
(nM)]
Ligand EPl EPz EP3 EP4 EP, EP, EP3 EP4
PGE, 9.1 4.9 0.33 0.79 20 12 0.85 1.9
SC19220* N.D. N.D. N.D. N.D. *** *** *** ***
Butaprost FA >10000 91 1643 >10000 *** 110 *** ***
Butaprost ME >10000 3513 r1oooo >10000 N.D. N.D. N.D. N.D.
Sulprostone 107 ** 0.35 7740 21 *** 0.6 ***
M&B28767 419 988 0.14 10 120 *** 0.68 500
GR63799 329 >10000 4.77 149 *** *** 1.9 480
Enprostil 82 >10000 12 >10000 *** *** *** ***
Misoprostol FA >10000 34 7.9 23 120 250 67 67
Misoprostol ME >10000 10249 319 5499 N.D. N.D. N.D. N.D.
11-deoxy PGE, N.D. N.D. N.D. N.D. 600 45 1.5 23
AH23848B* >10000 >10000 4419 13727 *** *** *** ***
PGD, 5280 2973 421 1483 *** *** 280 ***
BW245C >10000 219 r 10000 132 *** *** *** ***
ZK110841 1.8 6.0 -503 41 *** *** *** ***
BWA868C* N.D. N.D. N.D. N.D. *** *** *** ***
PGF,, 547 964 38 288 1300 *** 75 ***
Fluprostenol >10000 ** 708 >10000 *** *** *** ***
Cloprostenol 815 ** 4.4 9137 N.D. N.D. N.D. N.D.
Latanoprost 1750 >10000 6503 ** N.D. N.D. N.D. N.D.
Iloprost 11 1870 56 284 21 1600 22 2300
Cicaprost >I340 >I340 255 44 *** 1300 170 ***
Carbaprostacyclin 23 942 14 352 *** 1600 31 2300
U46619 >10000 >10000 >10000 2013 *** *** *** ***
SQ29548* ** ** ** ** *** *** *** ***
Human data from Abramovitz et al. (157). Mouse data from Kiryama et al. (164).
**K,estimate > 100,000 nM.
***Unableto displace 50% of radioligand at 10a.
over other EP receptors, making it a useful gastroduodenal damage and increased risk of
experimental tool. Like misoprostol it displays ulceration associated with NSAID use (233).
much higher affinity for recombinant EP, re- Examples include misoprostol, rioprostil,
ceptors when in its free acid form (116, 157). M&B28767, sulprostone, and nocloprost (84,
To date, there are no reports of compounds 234, 235). However, these compounds also
that act as specific EP, receptor blockers, al- seem to be active at other EP receptors. Miso-
though AH6809 is an antagonist at human prostol and rioprostil both have well-docu-
EP, receptors. A summary of the affinities and mented agonist activity at the EP, receptors,
potencies of EP, receptor ligands obtained in although not at EP, receptors, whereas sulpr-
both functional and binding studies are shown ostone, which has been widely used in EP, re-
in Tables 6.6 and 6.7, respectively. ceptor characterization studies, is also active
at EP, receptors (228). In contrast, enprostil
4.2.4 EP, Receptor-Selective Ligands. The and GR63799X are more selective for the EP,
structures of some ligands active at the EP, receptor (72, 236). In addition, both com-
receptor are illustrated in Fig. 6.7. EP, recep- pounds are highly potent in functional assays.
tors are believed to be important in mediating These functional assays have also been backed
the beneficial effects of prostaglandins in the up by the results of studies, both functional
stomach, where they seem to inhibit gastric and radioligand binding in recombinant recep-
acid secretion. Several EP, receptor agonists tors (116, 157, 164). Probably the most selec-
have been developed to protect against the tive agonist at the EP, receptor is SC46275
4 Prostanoid Receptor-Selective Ligands and Structure-Activity Relationships

AY23626

butaprost misoprostol
Figure 6.6. The structures of some EP, receptor-selective ligands.

(2371, which displays nanomolar potency in
smooth muscle preparations containing EP,
receptors, such as the guinea pig vas deferens,
but not those containing EP, or EP, receptors
(237).To date, there are no well-documented
at
m-
- are both weak EP, receptor antagonists, with
m a y of the affinities obtained in recom-
inant binding studies and functional poten-
for EP, receptor active compounds is
hown in Tables 6.6 and 6.8, respectively.
4.2.5 EP, Receptor-Selective Ligands. The
ented examples of potent, selective EP, re-
ptor agonists, but 11-deoxy-PGE, and
19R(OH)-PGE, have been identified as ago-
nists that display around 30-fold selectivity for
EP, receptors over the EP, receptor subtype
(116). However, recently, series of compounds
that show apparent EP, receptor selectivity
have been described (241, 242) The TP recep-
tor blocking drugs, AH23848B and AH 22921,
PA, values of around 5.4. Despite its low po-
tency, AH23848B is considered a relatively
specific antagonist (232). The structures of
some EP, receptor selective compounds, along
with their binding affinities and functional po-
tencies are shown in Fig. 6.8 and Tables 6.6
and 6.9, respectively.
4.3 FP Receptors and Selective Ligands
PGF,, is a potent FP receptor agonist, but it
also displays some activity at EP and TP re-

Equieffective Concentration
Relative to PGE, (= 1) Refs.
30-100 230
14 232
40 229
2-14 228
6-30 116,231
Note. there are no selective EP, receptor antagonists (1).
"Also has activity at EP, receptors.
bAlsohas activity at EP, receptors.
 Agents Acting on Prostanoid Receptors

Agonists
H

Sulprostone Misoprostol

Enprostil SC46275
Figure 6.7. The structures of some EP, receptor-selective ligands.

ceptors. PGF,, is a potent luteolytic agent and
is used in veterinary practice for this purpose.
Also potent luteolytics in several animal spe-
cies are two 16-phenoxy analogs of PGF,,,
known as fluprostenol and cloprostenol(244).
Their contractile actions on FP receptor prep-
arations, the iris muscle of the dog and the cat,
confirmed their identity as potent FP receptor
agonists. Of the two compounds, fluprostenol
is the most FP receptor selective, being almost
inactive at all the other prostanoid receptors,
whereas cloprostenol displays some weak ac-
tivity in EP, receptor-containing preparations
(232). Several other agents, all analogs of
PGF,,, have also been synthesized as FP re-
ceptor selective ligands. These include pros-

Table 6.8 Potencies of Some EP, Receptor Agonists

Equieffective Concentration
Agonists Relative to PGE, (=1) Refs.
Misoprostola 0.2-1.0 72,227,236
Rioprostol 0.9-1.1 72
M&B28767 0.1-0.6 74,227
Nocloprost 1 239
Enprostil 0.02-0.1 71, 72,236
Sulprostoneb 0.1-1.0 1,116,240
GR63799X 0.1 236
"Also has activity at EP, receptors.
bAlso has activity at EP, receptors.
4 Prostanoid Receptor-Selective Ligands
" and Structure-Activity Relationships

vn

11-deoxy-PGE,

AH23848B

Figure 6.8. The structures of some EP, receptor-selective ligands.

talene, fenprostalene, and tiaprost (245). In
addition, no specific FP receptor antagonists
have been reported. Several agents, 14-dide-
iF,,, N,N-dimethylamino-
and N,N-dime thy la mi do-^^&, have
ar whether these effects are mediated by FP
ceptor blockade (232). The structures of
Borne FP receptor ligands are shown in Fig. 6.9
and the binding affinities and functional po-
tencies are shown in Tables 6.4 and 6.10, re-
spectively.
Crossley
- (250) has investigated
- the struc-
ture activity requirements for FP receptor ac-
tivat,ion. Alteration of the length of the
p-chain, which can be altered from 8-11 car-
bon atoms, has little effect on activity. In ad-
dition, replacement of the carbon atoms in po-
sitions 17-21 in the p-chain with oxygen alters

Table 6.9 The Potencies of S o m e EP, Receptor Agonists and Antagonists

ve Concentration
Relative to PGE, (= 1) Refs.
11-deoxy-PGE, 1 116
19R(OH)-PGEZa 30 116
Antagonists P& Refs.

"Also has activity at EP, receptors.


CI
Cloprostenol
Figure 6.9. The structures of some F P receptor-selective ligands.
potency. The 17-keto derivative is the most
potent FP ligand, also displaying the greatest
selectivity over EP receptors. Furthermore,
replacement of the P-chain C-17-C-20 atoms
with phenoxy groups carrying further rneta
and para substitutions also affects FP recep-
tor interactions. The most potent FP receptor
agonist produced using this procedure is the
p-fluorophenoxy analog, but it also displayed
significant activity at TP receptors. However,
the rn-trifuoromethylphenoxy (fluprostenol)
and rn-chlorophenoxy (cloprostenol) substi-
tuted analogs are both highly potent and se-
lective FP receptor agonists (see above).
4.4 IP Receptors and Selective Ligands
The structures of some compounds with activ-
ity at IP receptors are shown in Fig. 6.10. PGI,
itself is chemically very unstable and is only
moderately selective, possessing agonist activ-
ity at EP, and TP receptors, as well as at IP
receptors (60). Several IP receptor ligands
Table 6.10 Potencies of Some FP Receptor Agonists
Equieffective Concentration
Agonists Relative to PGF,, (= 1)
Fluprostenol
Cloprostenol
Prostalene
Fenprostalene
Agents Acting on Prostanoid Receptors
Fluprostenol
Latanoprost
have been synthesized, with the aim of in-
creasing its stability and maintaining the po-
tentially beneficial platelet anti-aggregatory
actions of prostacyclin, but not its hypotensive
properties. The chemical instability of prosta-
cyclin stems from the close proximity of the
strained en01 structure and carboxylic group.
Hence, efforts to produce stable analogs have
focused on the substitution of the en01 ether
structure and also altering the length of the
a-side chain. Substitution of the oxygen in the
en01 group with less reactive sulphur, nitro-
gen, and carbon atoms has improved stability.
An example is carbaprostacyclin [(5E)-6a-
carba-PGI,] (251). To retain functional activ-
ity, it seems that the 1-carboxy and 11- and
15-hydroxy groups and unsaturation of C-5
are critical. Functional activity is enhanced by
the introduction of a methyl group at C-16 or
C-17. In addition, the presence of an alkyl
group at C-18 confers increased stability. This
feature is present in the first stable prostacy-
Refs.
0.2-1.0 246,247
0.3-0.5 248
0.7-2.7 248
1 249
4 Prostanoid Receptor-Selective Ligands and Structure-Activity Relationships

L
Carbaprostacyclin

lloprost Cicaprost

'I C02H

.
HO

Beraprost
Figure 6.10. The structures of some IP receptor-selective ligands.

clin analog described, iloprost, a molecule in
which the en01 ether oxygen present in PGI,
has been substituted with a methyl group and
the lower side-chain altered, including the in-
troduction of a triple bond at C-18, to improve
stability (252). A summary of the alterations
made to the structure of PGI, that increase
stability and potency at IP receptors is shown
in Fig. 6.11. Iloprost is as potent as prostacy-
clin in mediating the inhibition of platelet ag-
gregation and the relaxation of vascular
smooth muscle (2521, but it is not purely IP

Saturation of double bond
Replacement with C, N
-------
HO
Figure 6.11. Alterations that have been made to increase the stability of prostacyclin.
receptor-selective, having partial agonist ac-
tivity at EP, receptors (60). Therefore, cau-
tion must be used when interpreting func-
tional data from this compound in systems
known or suspected to contain EP, receptors.
However, the upper side-chain of iloprost is
susceptible to p-oxidation, resulting in a bio-
availabiltiy of only around 20% after oral dos-
ing (253). With this is mind, the same group
from Schering also produced cicaprost, which
is probably the most selective IP receptor ag-
onist currently available (254). In this com-
pound, they replaced the p-methylene group
at C-3 in the upper side-chain with an oxygen
atom to block the p-oxidation pathway, and
further altered the lower side-chain to pro-
duce this highly potent, stable compound.
These changes included the introduction of a
second triple bond at C-13 and an additional
methyl group at (3-20. It exhibits little func-
tional activity in EP receptor-containing
preparations (227) and is more potent than
both parent compounds at inhibiting platelet
aggregation and as a smooth muscle relaxant.
Cicaprost has also been suggested to have
anti-metastatic effects on tumors (253). Other
Table 6.11 Potencies of Some IP Receptor Agonists
Equieffective Concentration
Agonists Relative to PGI, (= 1)
Carbaprostacyclin
Iloprost
Cicapmst
Octimibate
"Primate IP, receptors only.
Agents Acting on Prostanoid Receptors
lntroduction of 0 at C-3
Replacement of double
bond with aromatic ring
Introduction of -CH3 at C-16
d
--
-
Extension of w-chain
analogs have also been described, including
beraprost, in which the en01 ether portion of
the PGI, molecule is incorporated into an ar-
omatic ring, and the lower chain is similar in
structure to that of iloprost. The affinities and
potencies of some IP receptor agonists are
shown in Tables 6.4 and 6.11, respectively.
In addition, several "non-prostanoid PGI,
mimetics" have been described as IP receptor
agonists. From the use of these agents, includ-
ing octimibate, EP157, and BMY45778, which
all lack a typical prostanoid ring, it has been
suggested that subtypes of IP receptor may
exist (19). To date, despite the array of selec-
tive IP receptor agonists, no specific antago-
nists have been described.
4.5 TP Receptors and Selective Ligands
The structures of some compounds that are
active at TP receptors are shown in Fig. 6.12.
TXA, itself is inherently unstable, having a
half-life in solution at pH 7.4 and 37°C of
around 30 s, making it unsuitable for rigorous
pharmacological studies. However, there are
several examples of stable TP receptor ago-
nists, the most useful of which are analogs of
Refs.
4 Prostanoid Receptor-Selective Ligands and Structure-Activity Relationships
PGH,. U44069 and U46619 (259) are 9,l l-ep-
oxymethano and 9,ll-methanoepoxy deriva-
tives of PGH,, respectively. These compounds
are the most widely characterized TP receptor
agonists in use today. U46619 is the best char-
acterized of the two ligands, and it displays
similar properties to TXA, in several prepara-
tions (104). In addition, it also causes platelet
aggregation and smooth muscle contraction
with similar potency to TXA, in vitro (260).
Other TP receptor agonists that have been
synthesized include EP171, SQ26655, I-BOP,
and ST& (32).
Agonists at TP receptors can be broadly di-
vided into two groups: those that are analogs
of TXA,, such as ST&, and those that are an-
alogs of PGH,, such as U46619. Replacing the
acetal moiety of TXA, not only stabilizes the
compound, but also may produce ligands that
display either agonist or antagonist proper-
ties. Hence, ST&, the thia analog of TXA,, is a
full agonist at TP receptors (261), whereas the
9(ll),lla-dicarba derivative, CT&, displays
partial agonist properties on human platelets
(262).Similarly, replacing the dioxygen bridge
in PGH, with isosteric moieties produces com-
pounds with TP receptor agonist activity. For
example, replacing either oxygen with a meth-
ylene group produces U44069 and U46619
(259).Furthermore, it seems that the incorpo-
ration of a p-fluorophenoxy moiety into the
terminus of the o-chain, as in EP171, in-
creases agonist potency.
Compared with the other prostanoid recep-
tors, TP receptors are unusual in that numer-
ous antagonists have been identified, several
of which are structurally unrelated to TXA,
itself. Some of the first identified drugs with
apparent TP receptor blocking activity in
blood platelets were the TXA, analogs, 9 , l l -
azoprosta-5,13-dienoic acid (263), 9,ll-epoxy-
iminod,l3-dienoicacid (264),and pinane TXA,
(265).However, these compounds all act as par-
tial agonists in smooth muscle preparations.
Further work produced compounds more dis-
tinct in structure from TXA,, such as the
7-oxabicyclo[2.2.l]heptane analog, SQ29548
(266), AH19437, AH23848B, and GR32191B
(vapiprost)(104, 172,267). EP045 and EP092
(263, 268) were generated as PGH, receptor
analogs. Compounds structurally unrelated to
prostanoids, such as BM 13505, which is a ben-
295
zylsulfonamido analog (2691,and the indole-2-
propanoic acid derivative, L-655240 (270),
have also been described as potent TP recep-
tor antagonists. Another potent, non-prostan-
oid TP receptor antagonist is BAYu3405 (271).
The affinities and potencies of some TP recep-
tor agonists and antagonists are shown in Ta-
ble 6.4 and Table 6.12, respectively.
4.6 Prostanoid Receptors: Location of Ligand
Binding Sites
Recent advances in molecular biology have
meant that the critical regions of amino acids
to confer high affinity ligand binding are now
beginning to be determined. For example, the
importance of arginine at position 329 in the
seventh transmembrane domain of all EP re-
ceptors has been examined by point mutations
in the rabbit EP, receptor. Thus, mutation of
this amino acid to alanine or glutamate abol-
ished the binding of [,HI-PGE,, whereas the
mutation of aspartate 388 was without effect
on ligand binding, but altered signal transduc-
tion, such that the EP, receptor agonist, sul-
prostone, was without effect even at high con-
centrations (279). Similar results have been
obtained using mouse receptors: mutation of
the equivalent arginine 309 to glutamatelva-
line produced a loss in ligand binding, whereas
switching it to lysine produced higher affinity
binding (280).These data suggest that the sev-
enth transmembrane domain of prostaglandin
receptors is involved in both ligand binding
and signal transduction.
Taken together with evidence that sug-
gests that modification of the carboxyl group
on the a-chain of prostaglandins tend to re-
duce agonist potency (219), the above findings
suggest that this conserved arginine could be
involved in interacting with the a-chain of
prostaglandin ligands. Indeed, it has been
shown that ligands containing methyl esters
at C-1 tend to display lower binding affinities
than those containing negatively charged hy-
droxy groups. An example is the comparison of
ligand binding of misoprostol methyl ester and
free acid to the EP, receptor (157). Further-
more, the rabbit EP, receptor displays a 370-
fold decrease in affinity for PGE, methyl ester
over PGE, (279). On the other hand, the same
authors demonstrated that sulprostone, which
contains a sulfonamide at C-1 that is bulkier

Agonists
. " ~ C O ~
OH
STA 2
Figure 6.12. The structures of some TP receptor-selective agonists.
than a carboxyl group but still carries a nega-
tive charge (pK, = 5.25 cf 5.19 for the carboxyl
group of PGE,), exhibited a threefold higher
affinity than PGE,. These data suggest that a
negative charge on C-1 is important for high
affinity ligand binding to EP receptors. Chang
and colleagues (281) have investigated the rel-
ative contribution of the conserved arginine
with respect to hydrogen bonding and ionic
interactions that may be involved in ligand
binding. They mutated the arginine to non-
charged but polar glutamine or asparagines,
or the non-polar leucine, to see how these
changes affected the binding of PGE,. The
mutation to leucine decreased binding affinity
by around 40-fold, whereas binding was al-
most unaffected by the other two mutations.
On the basis of this, they have suggested that
hydrogen bonding may be enough for high af-
finity ligand binding.
Agents Acting on Prostanoid Receptors
H
I-BOP
It is also interesting to speculate whether
the cysteine residue in the second extracellu-
lar loop forms a disulphide bridge that is im-
portant for receptor conformation. In the rab-
bit, mutation of this cysteine 204 to the
uncharged alanine did not affect PGE, bind-
ing (279). This is in contrast to results re-
ported in human TP receptors, where muta-
tion of the equivalent cysteine and another in
the first extracellular loop abolished ligand
binding (282, 283). Also, as the TP receptor
fails to display ligand binding after reduction
with dithiothreitol(284), and in other rhodop-
sin-like receptors equivalent cysteine residues
form a disulphide bridge, it has been sug-
gested that cysteine 105 and cysteine 183 may
form a disulphide bond essential to ligand
binding.
Studies with human TP receptors have also
highlighted several regions that are thought
4 Prostanoid Receptor-Selective Ligands and Structure-Activity Relationships

Antagonists

GR32191 (vapiprost)

L655240 BAYu3405
Figure 6.12. (Continued.)

to be important in ligand binding. For exam-
ple, the mutation of tryptophan 299, in the
seventh transmembrane domain, to leucine
produces a receptor that can bind the TP re-
ceptor agonist, U46619, but not the antago-
nist, SQ29548 (285). Furthermore, mutation
of the conserved arginine 295, also in the sev-
enth transmembrane domain of the human
TP receptor, causes a reduction in ligand bind-
ing (285). Likewise, the importance of cysteine
residues in the human TP receptor has also
been investigated by mutational studies,
298 Agents Acting on Prostanoid Receptors

Table 6.12 Potencies of Some TP Receptor Agonists and Antagonists

Equieffective Concentration
Agonists Relative to U46619 (= 1) Refs.
EP171 0.008-0.03 172,273
SQ26655 0.14-0.47 172
I-BOP 0.12-0.25 273,274
STA, 36 275
Antagonists Refs.

which have stressed the importance of those
residues in the first and second extracellular
loops (283). It also seems that glycosylation of
the TP receptor may be important for ligand
binding: when two extracellular glycosylation
sites are mutated, the resulting receptor dis-
plays a loss of ligand binding (286).
The use of chimeric receptors has also been
employed to attempt to dissect out regions of
receptors important in ligand binding. For ex-
ample, Kobayashi and colleagues (287) used
chimeric DP and IP receptors to study possible
domains involved in DP and IP receptor ligand
binding. In substituting different regions of
the mouse IP receptor with equivalent regions
from the mouse DP receptor, they suggested
that the sixth and seventh transmembrane do-
mains of the IP receptor confer specificity to
IP receptor ligands and that the third trans-
membrane domain of the DP receptor confers
the specific binding of PGD,. They suggested
that the IP receptor recognizes both the side-
chains and cyclopentane rings of the ligands it
binds. PGI, has a unique a-chain structure,
caused by the presence of an additional ring
attached to the cyclopentyl ring present in all
prostanoids. It has been suggested that the IP
receptor can recognize PGE,, but not PGE,,
because of the lack of the C-5-C-6 double
bond, which allows it to mimic the configura-
tion of the side-chain in I series prostaglan-
dins. Likewise, the binding of I- and E-series,
but not D- and F-series prostaglandins, was
explained by the lack of cyclopentane ring rec-
ognition for the latter two prostaglandin se-
ries. Kedzie and co-workers (288) employed a
similar approach, mutating EP, receptor res-
idues common to EP, and EP, receptors, but
not IP receptors to those present in IP recep-
tors. They showed that mutating the human
EP, receptor leucine 304 in the seventh trans-
membrane domain to tyrosine resulted in an
increase in potency of iloprost of around 100
times in a CAMP-dependent reporter gene as-
say. Likewise, mutation of the critical arginine
302 resulted in a loss of potency for all prosta-
noids tested. This may be consistent with the
above assertion that amino acids in the sixth
and seventh transmembrane domains are rel-
evant in the binding of the a-chain of IP li-
gands. Hence it can be seen that some progress
is being made into the regions of prostan-
oid receptors that confer high affinity ligand
binding.
5 CLINICAL USE OF AGENTS
The current drugs available in the clinic are
mostly natural prostaglandins themselves and
a few closely related analogs, and it is only
very recently that efforts have again increased
to find highly selective synthetic prostaglan-
din receptor ligands, which might be useful
medicines. One barrier to this effort, at least
with respect to pain and inflammation, is that
aspirin and the competitive COX inhibitors,
which inhibit prostaglandin synthesis per se,
are already available and have proved to be
remarkably efficacious. However, they do
cause unwanted gastrointestinal side effects,
especially the non-selective COX inhibitors. It
5 Clinical Use of Agents
remains to be seen whether selective prosta-
noid receptor antagonists might provide bet-
ter medicines for the treatment of pain and
other therapeutic indications. A summary of
the available agents and their indications is
presented in Table 6.13. The therapeutic areas
in which these agents are used are diverse and
are a consequence of the widespread physio-
logical actions of the natural prostaglandins.
Prostaglandins used in the termination of
pregnancy, cervical ripening, and labor induc-
tion include dinoprost (PGF,,) and carbaprost
(15-methyl-PGF,,), dinoprostone (PGE,), sul-
prostone, gemeprost, and misoprostol. Alpros-
tadil (PGE,) is used to treat erectile dysfunc-
tion. Prostaglandins used in cardiovascular
conditions include alprostadil and the PGE,
analog, limaprost, as well as epoprostenol
(prostacyclin) and its analog, iloprost. Miso-
prostol is used clinically, with some success, as
an inhibitor of gastric acid secretion. Prosta-
glandins, particularly of the F series, are
widely used as luteolytics in veterinary medi-
cine, but a discussion of this is beyond the
scope of this chapter. However, the FP recep-
tor agonist, latanoprost, has found use in the
treatment of glaucoma (289, 290). D-series
prostaglandins are not used in the clinic at
present. Intravenous administration of PGD,
in humans causes facial flushing and intense
nasal congestion. No effects were seen with
respect to ADP-induced platelet aggregation,
suggesting PGD, is unlikely to be a useful
anti-thrombotic in man (291). Each clinically
available prostanoid is described below, begin-
ning with the natural prostaglandins and then
the selective ligands that have been developed
with the aim of producing more stable com-
pounds, with a longer duration of action and a
more specific effect. At present, the clinically
available prostanoids are all receptor agonists.
The structures of some of the clinically avail-
able prostanoids not covered in Section 4 are
shown in Fig. 6.13.
5.1 Natural Prostaglandins
5.1.1 Alprostadil (Prostaglandin E,). Alpro-
stadil is the name given to an injectable for-
mulation of PGE,, which when administered
exogenously causes vasodilatation and the in-
hibition of platelet aggregation. It exerts its
\R 3 %@%J
actions primarily through - EP and IP prosta-
noid receptors. The main clinical usesare in
the treatment of male erectile dysfunction and
congenital heart disease. Alprostadil is largely
inactivated by the lungs on its first passage
through the pulmonary circulation. Its metab-
olites are excreted in the urine within approx-
imately 24 h.
Alprostadil is available in several formula-
tions for the diagnosis and treatment of impo-
tence caused by erectile dysfunction. The most
common formulation is for intracavernosal in-
jection (5-20 pg). For intracavernosal injec-
tions, it is recommended that the dose given
does not exceed 60 pg, although doses of up to
100 pg have been reported. Dose-related re-
sponses have been reported (292). Following
intracavernosal alprostadil injection, the inci-
dence of pain at the injection site has been
reported to be dose-dependent (293). In addi-
tion, penile fibrosis and priapism has also been
observed when prostaglandins are used for
this purpose. In cases of priapism, corrective
therapy should be administered rapidly. In
comparative studies, intracavernosal alpros-
tadil was as effective at producing erections as
papaverine alone or in combination with the
non-selective a-adrenoreceptor antagonist,
phentolamine (294, 295). It has also been ad-
ministered in combination with these two
agents. Other administration routes include
urethral suppositories, but the dose required
is higher (125-1000 pg rather than the usual
dosing range of between 5 and 20 pg) (296). In
a recent trial, intraurethral alprostadil was re-
ported to have an overall success rate of
around 50% (297).
Alprostadil is also used to treat several as-
pects of cardiovascular disease. For example,
it is administered intravenously to neonates
with congenital heart disease to maintain the
patency of the ductus arteriosus before sur-
gery can take place. In neonates treated with
alprostadil for congenital heart defects, sev-
eral side effects were observed in a 3-year
study examining almost 500 children. These
included some cardiovascular events, and in
some individuals, respiratory depression
(298). Alprostadil has also been used in the
treatment of peripheral vascular disease, es-
pecially Raynaud's syndrome, which is charac-
terized by digital ischemia induced by cold and
300 Agents Acting on Prostanoid Receptors

emotion (299). In addition, several studies mainstream therapies. Alprostadil is one of

have shown it to be superior to placebo in pe- several drugs that have been used to combat
ripheral arterial occlusive disease (PAOD) circulatory disturbances in ergotamine poi-
(300). However, such uses do not constitute soning (301) and has also proved effective in

Table 6.13 Agents in Clinical Use That Are Active at Prostanoid Receptors
Generic Trade
Name Name(s) Originator Synonyms Dose
Alprostadil Caverject Pharmacia PGE, Ductus arteriosus:
Upjohn 50-100 ng kg-'
min- '
Erectile dysfunction:
5-20 pg
intracavernosally
Muse AstraZeneca Erectile dysfunction:
125-1000 pg
intraurethrally
Dinoprostone Prepidil Pharmacia PGE, Cervical ripening:
Upjohn 500 pg in 2.5 ml
(gel),
Labour induction:
1-2 mg per 2.5 ml
(gel), 0.5 mg h-'
orally
Prostin Pharmacia Termination of
E2 Upjohn pregnancy: 100 pg
intra-amniotically,
5 pg ml-I IV
Limaprost Opalmon On0 ONO-1206 Anti-thrombotic
Gemeprost Cervagem Rhone- 16,16 Cervical ripening: 1
Poulene dimethyl mg pessary
PGE, vaginally
Misoprostol Arthotec Searle SC-29333 Anti-ulceration: 800
orally
Cytotec Searle Labour induction:
25-50 pg vaginally
Sulprostone Nalador Schering AG CP-34089; Cervical dilation:
SHB 500 pg per 3-6 h
286; ZK- IV
57671 Termination of
pregnancy: 100-
500 pg h-' IV
Latanoprost Xalatan Pharmacia PhXA-41; Glaucoma: 0.005%
Upjohn XA-41 solution topically
Epoprostenol Flolan GlaxoSmithKline Prostacyclin; Dialysis: 5 ng kg-'
PGI, min-' IV
Pulmonary
hypertension:
continuous
infusion after
dose-ranging
Iloprost Ilomedin Schering AG ZK-36374 Peripheral vascular
disease: 0.5-2 ng
kg-I IV as
trometamol salt
5 Clinical Use of Agents 301

Table 6.13 (Continued)

Trade
Narne(s) Originator Synonyms Dose
Hemabate Pharmacia 15-methyl Termination of
Upjohn PGF2,; pregnancy: 250-
U-32921 500 pg IM
Prostinfenem Pharmacia Postpartum
Upjohn haemorrhage: 250
Pg IM
Prostin Pharmacia PGF2, Termination of
Pregnancy: 40 mg
intra-amniotically,
25-50 pg min-'
IV
Labour induction:
2.5-20 pg min-'
IV
Prostarmon Ono Ileus: 0.3-0.5 ~g
F kg-' min-' IV

the management of hemorrhagic cystitis in
children following bone marrow transplanta-
Alprostadil also seems to be beneficial in
the treatment of some hepatic disorders, with
some promise being shown in patients with
fulminant or subfulrninant viral hepatitis
(303). In these cases it has also been adminis-
red with dinoprostone and the EP, and EP,
ceptor agonist, misoprostol.
In addition to the side effects outlined
ove, administration of exogenous PGE, may
produce some irritation at the infusion site,
headache, and vasodilation. The vasodilatory
effects also occur with prostacyclin analogs
and are often more pronounced in these cases.
The rare occurrence of gastric mucosal hyper-
plasia has also been reported in children re-
ceiving long-term treatment with prostaglan-
dins (304).
Dino-
5.1.2 Dinoprostone (Prostaglandin E,).
prostone is the name given to pharmaceutical
formulations of PGE,, and the action most ex-

HO

+-
' CO~H

.
0
Gemeprost Unoprostone

Limaprost
Figure 6.13. The structures of some compounds in clinical use that are active at prostanoid

ploited in the clinic is its ability to contract
smooth muscle. It exerts its actions predomi-
nantly through interactions with EP recep-
tors. It is widelv
" used in the induction of labor
but also for the termination of pregnancy, hy-
datiform mole, and missed abortion. The main
route of administration is vaginal, but this
drug can also be dosed intravenously, orally or
extra-amniotically. Intravenous infusion is as-
sociated with a large incidence of adverse ef-
fects (305).
In labor induction, dinoprostone is used to
soften and dilate the cervix before the mem-
branes are breached and labor is induced. It
may also be administered either vaginally or
orally to induce labor. However, the oral route
is not common, because it is associated with a
greater incidence of gastrointestinal adverse
effects. Likewise, intravenous dosing has also
been employed, but this is no longer generally
recommended.
Dinoprostone is also used in the termina-
tion of pregnancy in the second trimester. For
this purpose, it is usually dosed extra-amnioti-
cally through a suitable catheter, with the
dose given being dependent on patient re-
sponse. Intravenous infusions have also been
used for this purpose, as well as in cases of
hydatiform mole and missed abortion. In the
United States, the preferred route of adminis-
tration for this purpose is through vaginal pes-
sary. Although they are effective alone, be-
cause of the incidence of adverse effects,
prostaglandins are normally used at low doses
in combination with mifepristone (306).
Along with alprostadil (see above), dino-
prostone is also used in the short term to
maintain ductus arteriosus patency before
surgery. Oral dinoprostone, when used longer,
may be beneficial in allowing later surgery by
facilitating infant growth. This has been re-
ported by both Silove and colleagues (307) and
Thanopoulus and co-workers (308) in 60 and
22 infants, respectively. Initial treatment reg-
imens typically continue for 4 weeks, and dos-
ing is typically oral, but can be intravenous if
gastrointestinal absorption is poor.
Like alprostadil and other prostaglandins,
dinoprostone
. has also been used in the man-
agement of hemorrhagic cystitis, especially
that caused by cyclophosphamide (309),and in
cardiovascular disease, including Raynaud's
Agents Acting on Prostanoid Receptors
syndrome. However, the latter therapy is not
widely used. Furthermore, dinoprostone has
proved useful in treating the oral lesions in
patients with pemphigus vulgaris.
Side effects occurring following dinopros-
tone seem to be related to the dosing system
used, and the intravenous route is associated
with more adverse reactions. Common prob-
lems include nausea, diarrhea, and abdominal
pain, along with short-term cardiovascular ef-
fects such as headache, hypotension, and fa-
cial flushing, as seen with alprostadil. How-
ever, a report cataloguing 3313 pregnancies in
which dinoprostone was used in cervical rip-
ening or labor induction reported that side ef-
fects were rare and were generally limited to
diarrhea, vomiting, and fever (310). The most
common adverse effect seen with the use of
prostaglandins combined with mifepristone is
excessive vaginal bleeding.
5.1.3 Dinoprost (Prostaglandin F,,). This
is the name given to pharmaceutical formula-
tions of PGF,, and is widely used in the ter-
mination of pregnancy, because it exerts a
contractile effect on uterine smooth muscle at
any stage of the gestation period. However, it
is also used, like dinoprostone, for missed
abortion, hydatiform mole, and fetal death in
utero. It is no longer generally recommended
for routine use in the induction of labor be-
cause of the higher incidence of adverse effects
encountered compared with dinoprostone.
For termination of pregnancy, the intra-
amniotic route is preferred, because this re-
sults in fewer side effects than intravenous
dosing. Dinoprost has also been used success-
fully to treat the ileus that results from vinca
alkaloid administration (311).
Dinoprost produces similar adverse effects
to dinoprostone, and epoprostenol and ilo-
prost have also both been reported to produce
similar cardiovascular effects (312).
5.1.4 Epoprostenol (Prostaglandin I,; Pros-
tacyclin). Epoprostenol is the name used to
describe a prostacyclin formulation for exoge-
nous administration. This drug has a very
short half-life in the body and is rapidly bro-
ken down to 6-keto-PGF,,. Prostacyclin is a
vasodilator and potent inhibitor of platelet ag-
gregation and is used to treat pulmonary hy-
5 Clinical Use of Agents
pertension and to prevent platelet aggregation
in, for example, blood from patients undergo-
ing kidney dialysis. Epoprostenol has also
been evaluated for the treatment of heart fail-
ure. However, long-term use resulted in an
increase in patient mortality, so development
for this purpose has now been discontinued
(313). Like alprostadil and dinoprostone,
epoprostenol has also been used in the treat-
ment of peripheral vascular disease.
Owing to its instability, epoprostenol is
supplied as the sodium salt and must be ad-
ministered by continuous infusion. As a con-
sequence, a dose-ranging study must first be
performed to evaluate the maximum tolerable
infusion rate. Epoprostenol has also been used
with some success in patients with acute respi-
ratory distress syndrome (314, 315).
In pulmonary hypertension, epoprostenol
was first introduced as a short-term treatment
enabling patients to undergo heart-lung trans-
plants. However, in some patients treated long
term with the drug, an encouraging clinical
improvement has been reported (316-318).
The intravenous route of administration is
difficult to manage because it requires contin-
uous infusion, but as an alternative, inhala-
tion of epoprostenol may provide a therapy
with fewer adverse effects, and this regimen
has been used successfully in adults with both
primary and secondary pulmonary hyperten-
sion (319, 320). Epoprostenol has also been
ed in organ transplantation, finding use in
th organ preservation and also in the treat-
nt of some of the post-transplant complica-
ons that may occur.
Epoprostenol has also been used in patients
h ischemic stroke. However, the patho-
hysiological involvement of endogenous
rostaglandins in ischemic brain damage is
ot clear, and no studies showing significant,
stained therapeutic benefits with epoproste-
01 have been reported (321,322).
5.2 Synthetic Prostaglandins
5.2.1 Gemeprost. Gemeprost is a synthetic
analog of PGE,. It is usually given vaginally as
pessaries and is used therapeutically to soften
and dilate the cervix and to stimulate uterine
moot4 muscle contraction in the termination
of pregnancy. For the latter indication, ge-
meprost may be given intravaginally in con-
juction with oral mifeprostone (306). Mifepro-
stone is a progesterone antagonist and serves
to sensitize the uterus to prostaglandins. This
facilitates the use of lower doses of prostaglan-
dins in pregnancy termination, thus reducing
the severity and incidence of unwanted ad-
verse effects. However, gemeprost is expen-
sive and is thermolabile, requiring refriger-
ated storage. It is not approved for use in the
United States (306).
Side effects following intravaginally ad-
ministered gemeprost are generally relatively
mild, with systemic effects such as nausea and
diarrhea reported. The effects of this drug on
the fetus are not known, but once a prosta-
glandin has been used to initiate termination
of pregnancy, if it is found to be unsuccessful,
termination must be completed by another
means.
5.2.2 Misoprostol. Misoprostol is a syn-
thetic analog of PGE, and is regarded as a
selective EP, and EP, receptor agonist. It has
a higher affinity at the EP, receptor, and this
has been demonstrated in recombinant sys-
tems using the cloned receptors of several spe-
cies (157, 164). It is a mixture of four stereo-
isomers, with the llR,16S isomer accounting
for most of its activity, and it will be interest-
ing to see if stereochemically pure formula-
tions show greater therapeutic efficacy. Once
administered, it is rapidly metabolized to its
active form, misoprostol-free acid. It is further
metabolized by oxidation in several organs
and is excreted mainly in the urine. It has a
plasma elimination half-life of around 20-40
min (323) and is about 85% serum albumin
bound (324).
Misoprostol has several therapeutic uses,
including the treatment of gastroduodenal ul-
ceration and labor induction and has the ad-
vantage of being relatively inexpensive and
stable enough to be stored at room tempera-
ture. Prostaglandin therapy for the induction
of labor is a well-established practice, and mi-
soprostol has been compared with dinopros-
tone and also oxytocin. It was found to be
equieffective with oxytocin (325) and more ef-
fective than dinoprostone (326). It was also
shown to be effective in 92% of women in com-
bination with tamoxifen (327). However, mi-

soprostol is often not effective in the termina-
tion of pregnancy when used alone. Its misuse
has led to reports of associated congenital ab-
normalities in neonates, including scalp, skull,
and limb defects. It is possible that these ter-
atogenic effects may be caused by an increase
in uterine pressure caused by vascular spasm
or uterine contractions (328, 329). However,
no such events have been reported in animal
studies (330). Misoprostol has also been
shown to be effective in treating both gastric
and duodenal ulcers but is no more efficacious
than other established therapies, such as his-
tamine H2-receptorantagonism, and has more
unwanted side effects (331, 332). One condi-
tion where it was thought it might offer supe-
riority over standard antacid therapy was in
ulcer prevention in patients on long-term
NSAID treatment (333). Nevertheless, proton
pump inhibitors, such as omeprazole, seem to
be as effective and have fewer side effects.
Misoprostol may also prove useful in the
treatment of postpartum hemorrhage and has
also been used in organ transplantation. For
example, it has been reported to improve kid-
ney function in cyclosporin-treated patients
who have received renal transplants (334).
The most common adverse effect seen with
the use of misoprostol is diarrhea, but abdom-
inal pain has also been reported, limiting the
use of this and other similar compounds in the
management of gastroduodenal ulceration.
Effects on uterine contraction have also been
reported, suggesting that misoprostol should
not be given to pregnant women (335).
5.2.3 Sulprostone. Sulprostone is a syn-
thetic derivative of PGE, and is primarily rec-
ognized as an EP, receptor agonist, although
it also has activity at EP, receptors (116). It
was developed as an abortifacient, and like
other PGE analogs, it acts as a uterine stimu-
lant. It has thus been used to stimulate cervi-
cal dilation before pregnancy termination dur-
ing the first 3 months of gestation. Dosing is
generally intravenous, although several other
routes have been used, including intramuscu-
lar, although this is no longer recommended.
It has also been used in the treatment of post-
partum hemorrhage. Sulprostone's use as an
abortifacient has been discontinued, because
Agents Acting on Prostanoid Receptors
of a link with increased cardiovascular compli-
cations, such as hypotension and acute myo-
cardial infarction (336). Sulprostone has a
similar side effect profile to dinoprostone.
5.2.4 Latanoprost. Latanoprost is a deriva-
tive of PGF,, that is used in the treatment of
glaucoma and ocular hypertension, and in-
creases the outflow of the aqueous humor, by-
passing the obstructed site of normal drainage
by opening an alternative drainage pathway
through uveal and scleral tissues. It is used
topically as eye drops, and as an adverse effect,
it may induce an increase in brown pigmenta-
tion of the iris, corneal deposits, and, rarely,
an increase in eyelash growth. This effect is
caused by increased melanin formation in me-
lanocytes. In addition, latanoprost also pro-
duces increased reductions in intraocular
pressure when used with other anti-glaucoma
agents, such as the muscarinic agonist, pilo-
carpine, and the carbonic anyhydrase inhibi-
tor, acetazolamide (337). It has the advantage
that it only requires once daily administra-
tion, thus improvingpatient compliance (338).
Unoprostone is another PGF,, analog that
has also been used in the treatment of glau-
coma, although mainly in the Japanese mar-
ket (339).
5.2.5 Iloprost. Iloprost is a stable analog of
prostacyclin, and like PGI,, causes vasodila-
tion and the inhibition of platelet aggregation.
In addition to acting at IP receptors, it dis-
plays potent partial agonist activity at EP, re-
ceptors (60). It has found clinical use, along
with other prostaglandins, in the treatment of
peripheral vascular disease and several ad-
ministration routes have been investigated for
its use in the management of pulmonary hy-
pertension, a disease where epoprostenol is an
established therapeutic (318). A recent, un-
controlled trial has suggested that inhaled ilo-
prost may provide a novel treatment option in
patients with life-threatening pulmonary hy-
pertension that is unresponsive to current
therapies (340). In particular, it has been used
to combat pulmonary arterial obstructive dis-
order, which tends to occur following artery
narrowing and occlusion influenced by athero-
sclerosis. This therapy is usually well toler-
References
ated, with headache and facial flushing being
the most common adverse effects (341).
5.2.6 Carboprost. Carbaprost is a 15-
methyl analog of dinoprost (prostaglandin
F,,) and is also known as methyldinoprost.
Like dinoprost, it is a uterine stimulant, but
because of the presence of the 15-methyl
group, it has a more prolonged course of ac-
tion, because this delays enzymatic dehydro-
genation associated with inactivation.
Clinical use is generally for the termination
of pregnancy, but it is also used in postpartum
hemorrhage. Following administration to the
bladder, it has also proved useful in cyclophos-
phamide-induced hemorrhagic cystitis in bone
marrow patients (342).
5.2.7 Limaprost. Limaprost (ONO-1206)is
a synthetic prostaglandin E, analog that has
been used in the treatment of peripheral vas-
cular disease. It is usually administered orally.
Compared with PGE,, limaprost has two ad-
ditional methyl groups at C-17 and C-20 and
also an extra double bond at C-2-C-3. It has
also been shown to have some efficacy in the
treatment of Raynaud's syndrome (343).
6 NEW DEVELOPMENTS
All of the clinically useful prostaglandins at
present exert their actions as agonists at pro-
stanoid receptors, and are in general, synthet-
ically produced natural prostaglandins or
closely related analogs. Hence, although there
has been a trend towards the exploitation of
prostanoid receptor subtype specific ligands as
potentially useful medicines, the compounds
that have been used to date have failed to fulfil
their therapeutic promise. For example, it was
hoped that EP, receptor agonists, such as
GR63799X and misoprostol, might prove ben-
ficial as cytoprotective agents, but they
roved to be less effective, in this respect, than
tamine H, receptor antagonists, such as ra-
However, the recent success of both la-
tanoprost and unoprostone in the treatment
f glaucoma (338, 339) suggests that FP re-
eptor agonists a t least may have an impor-
tant future role in the clinic. Recent evi-
305
dence from knock-out mouse studies has
further suggested the potential involvement
of EP,, EP,, and IP receptors in mediating
painful responses (67, 344). Coupled with
this, the use of EP, receptor antagonists for
the treatment of inflammatory pain has re-
cently been suggested (63). In light of the
possibility that DP and EP, receptor antag-
onists might prove useful in the treatment of
allergic inflammation and migraine, respec-
tively (2 11,345),there is evidence to suggest
that, in the future, potent specific prosta-
noid receptor antagonists may make effec-
tive medicines.
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 gon State University
oratory of Molecular Pharmacology
epartment of Pharmaceutical Sciences
ollege of Pharmacy

Contents
1 Introduction, 318
2 Clinical Applications, 319
3 Retinoid Biosynthesis, 320
3.1 Fate of Dietary Sources of Retinoids, 320
3.2 Symmetrical Cleavage of p-Carotene, 321
3.3 Asymmetrical Cleavage of /%Carotene, 322
3.4 Formation of Retinyl Esters, 323
3.5 Mobilization of Body Vitamin A Stores, 324
3.6 The Fate of Intracellular Retinol, 324
3.7 Oxidation of Retinol, 325
3.8 Formation of Retinoic Acid: A Major
Signaling Retinoid, 325
3.9 Active Isomers of q 3 2 6
3.10 14-Hydroxy-4,14-retro-retinol, 327
3.11 Non-Receptor, RA Binding Proteins, 328
4 Retinoid Metabolism, 328
5 Role of Retinoids in Visual Signal Transdudion,
330
5.1 Photon Detection, 331
5.2 Initiation of a Visual Signal, 331
5.3 Shutting Off R*,331
5.4 The Retinoid Cycle in Visual Processes, 332
5.5 Retinoids and the Basis of Color Vision, 334
6 Retinoic Acid Signaling Pathways, 335
6.1 Historical Perspective, 335
6.2 Discovery of RXRs, 336
6.3 Identification of Retinoic Acid Target Genes,
337
6.4 RARs and RXRs Bind Response Elements as
a Heterodimeric Complex, 339
7 Functional Domains of RARs
Burger's Medicinal Chemistry and Drug Discovery and RXRs, 340
Sixth Edition, Volume 4: Autocoids, Diagnostics, 7.1 RAR and RXR DNA Binding Domains, 340
and Drugs from New Biology 7.2 Ligand Binding Domain, 341
Edited by Donald J. Abraham 7.3 Apo-Receptors Interact with Transcriptional
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc. Corepressor Complexes, 341

7.4 Holo-RAR.RXRComplexes Interact with
Two Classes of Transcriptional Coactivators,
344
7.5 "Ligand-Independent" Transcriptional
Activation, AFT, of RARs and RXRs, 345
8 Future Perspectives, 345
8.1 Receptor- and Receptor Subtype-Selective
Retinoids, 345
1 INTRODUCTION
Retinoids are derivatives of vitamin A that
have both diverse and essential actions in
developmental and cellular differentiation
processes, vision, and reproduction (1). The
biological activity of retinoids has been appre-
ciated since the 1920s (2, 3), and the huge
strides made by synthetic chemists, and mo-
lecular, developmental, and structural biolo-
gists since the late 1980s have contributed
dramatically to our understanding of the mo-
lecular processes that underlie the biological
activities of retinoid compounds. Retinoids are
now well established as valuable therapeutic
agents in the treatment of a variety of skin and
proliferative disorders. Indeed, the number of
retinoid compounds approved by the Food and
Drug Administration (FDA) for clinical use,
seven, has more than tripled since the last edi-
tion of this text, and there are no indications
that the wave of retinoid drug development
has reached a crest.
Figure 7.1. Naturally occurring
trans-retinoids.
Retinoids
8.2 "Gene-Specific" Retinoids, 348
8.3 Anti-AP1 Activity of Retinoids, 349
8.4 CD437: An Atypical Retinoid, 350
8.5 Novel Uses for RXR-Active Compounds, 350
8.6 Summary and Perspective, 351
Naturally occurring retinoids are charac-
terized by three distinct structural elements
(Fig. 7.1): (1)a lipophilic terminus composed
of a p-ionone ring, (2) an isoprene side chain
that is subject to both enzymatic and non-en-
zymatic isomerization (Fig. 7.2), and (3) a po-
lar terminus that is the subject of oxidative
modifications (Figs. 7.1 and 7.2). All of these
processes occur in vivo with the superimposed
complexity of a multitude of binding and
transport proteins and receptors possessing
differential affinities for these naturally oc-
curring retinoids.
The objectives of this chapter are to describe
(1)retinoids that are currently in clinical use,
including the adverse effects associated with
the therapeutic use of these compounds; (2)
retinoid biosynthesis and metabolism; (3) the
basis of action of retinoids in molecular, cellu-
lar, and organismal contexts including vision;
and (4) new agents on the horizon of retinoid
biology.
Isoprene Polar
side shain terminus
C02H trans - retinoic acid
2 Clinical Applications

CHO

9-cis-retinoic acid

ecO
2 CLINICAL APPLICATIONS
13-cis-retinoic acid Figure
noids. 7.2. Naturally occurring cis-reti-

fulness of these compounds. Systemic retinoid

com~oundsare considered to be extreme te-
A

Early studies conducted by Wolbach and ratogens, inducing severe malformations of

Howe suggesting that vitamin A plays a key the central nervous system, skull, external
role in limiting cellular proliferation and in ear, eye, cardiovascular system, thymus, and
the maintenance of the terminally differenti- parathyroid gland. (Table 7.2 describes terato-
ated phenotype of a number of cell types (2,3) genicity associated with systemic isotretinoin
set the stage for the present use of naturally use.)
occurring retinoids and synthetic derivatives Owing to the extreme teratogenicity of sys-
in the treatment of diseases of the skin (psori- temic retinoid compounds, the FDA and
asis, severe acne, and photodamage) and pro- Roche Pharmaceuticals have taken stringent
liferative disorders (acute promyelocytic leu- measures to prevent pregnant women from
kemia, cutaneous T cell lymphoma, and being administered systemic isotretinoin and
Kaposi's sarcoma). Retinoid compounds pres- to prevent women taking isotretinoin from be-
ently possessing FDA approval for clinical use coming pregnant. All other systemic retinoid
are listed in Table 7.1 and structures are compounds bear similar warnings. Well-con-
shown in Fig. 7.3. In addition, the reader is trolled studies on the potential human terato-
advised to consult governmental (http:l/ genicity of topically applied retinoids have not
clinicaltrials.gov/) and Pharmaceutical Re- been conducted; however, common sense dic-
search and Manufacturers of America (http:ll tates that topical use of retinoids during preg-
www.phrma.org/searchcures/newmedslwebdb/ nancy should be avoided.
drugs.phtm1) web pages that indicate all on- Non-teratogenic, adverse effects of retin-
going clinical trials that may ultimately lead to oids are also severe and are dependent on
the use of retinoids to treat a wider spectrum route of administration and the nature of the
of diseases including cancers of the skin, disease being treated. Untoward effects of ret-
breast, kidney, bladder, prostate, and head- inoid compounds administered by mouth are
and-neck, neuroblastomas, acute myeloid leu- similar to those of hypervitaminosis A and in-
kemia, Hodgkin's lymphoma, and emphy- clude cheilitis and pronounced lipid distur-
sema. bances such as hypertriglyceridemia, hyper-
Adverse effects of retinoids in current use cholesterolemia,and reduction in high density
are generally severe and limit the clinical use- lipoprotein levels, pancreatitis, vasculitis, con-
320 Retinoids

Table 7.1 Retinoids Currently Approved by FDA for Clinical Use

Route of
Generic Name Trade Name Administration Therapeutic Use
Adapalene Differin Topical Acne vulgaris
Acitretin Soriatane Topical Psoriasis
Alitretinoin Panretin Topical Kaposi's sarcoma
(9-cis-retinoic acid)
Bexarotene Targretin Topical Cutaneous T-cell
lymphoma
Isotretinoin Accutane Topical Severe acne
(13-cis-retinoic Oral Severeacne
acid)
Tazarotene Tazorac Topical Psoriasis
Tretinoin (trans- Renova Topical Skin photodamage
retinoic acid) including
wrinkles, sun
spots, and
roughness
Retin-A Topical Severe acne
Vesanoid Oral Acute
promyelocytic
leukemia
Solange (a combination Topical Solar (actinic)
of tretinoin and lentigines
mequinol, a
depigmenting agent)

junctivitis, edema, lymphadenopathy, weight administration, including irritation as indi-

loss, GI disturbances including inflammatory
bowel disease, dysregulation of blood glucose
levels, benign intracranial hypertension,
hearing impairment, fatigue, depression, de-
calcification of bone, calcification of cartilage
and tendons, hair loss, and skin scaling.
Adverse reactions associated with use of
the systemic administration of tretinoin We-
sanoid) in the treatment of acute promyelo-
cytic leukemia (APL) are particularly severe,
resulting in what has come to be known as
"Retinoic Acid-APL Syndrome." Approxi-
mately 25% of APL patients treated with
tretinoin develop this syndrome, which is
characterized by fever, dyspnea, weight gain,
pulmonary infiltrates, and pleural or pericar-
dial effusions resulting in compromised myo-
cardial function and episodic hypotension. In
addition, approximately 40% of patients un-
dergoing oral tretinoin therapy for APL de-
velop a rapidly evolving leukocytosis that can
be associated life-threatening complications.
Topically administered retinoids, although
generally free of systemic toxicity, cause mod-
erate-to-severe adverse reactions at the site of
cated by development of a rash (erythema,
scaling, irritation, redness, dermatitis), dry-
ness, burning, pruritis, peeling, blistering,
and edema. In many cases, initiation of topical
retinoid therapy results in an exacerbation of
the disease, such as acne, for which the reti-
noid is being used. This effect is generally tem-
porary and subsides with continued therapy,
but in severe cases, requires discontinuation
of the retinoid agent.
3.1 Fate of Dietary Sources of Retinoids
Vitamin A is essential for life, and although
humans and other animals are capable of mod-
ifying retinoid compounds, these species are
incapable of the de novo synthesis of retinoids.
Thus, dietary intake of the retinoids is crucial
for the maintenance of health and prevention
of vitamin A deficiency. Dietary retinoids con-
sist of two general types: (1)carotenoids, in-
cluding principally p-carotene as well as po-
tentially hundreds of other related compounds
 3 Retinoid Biosynthesis

Bexarotene
0

0CH2CH3

lsotretinoin
(13-cis-RA) Tazorotene

Tretinoin
(trans-RA)

Figure 7.3. Chemical structures of FDA-approved retinoids.

that are derived primarily from plant sources,
and (2)several forms of retinyl esters that are
derived from the consumption of other ani-
mals. Both of these precursors give rise to ret-
in01 that is the most abundant retinoid in
blood, whereas retinyl esters in liver and other
tissues represent the major storage form of
retinoids in the body.
3.2 Symmetrical Cleavage of p-Carotene
i substantially clarified this area. These break-
1
: The small intestine is a major site of retinoid
j metabolism, beginning with the oxidative
I
cleavage of p-carotene into two molecules of
retinaldehyde (Fig. 7.4). The subject of p-car-
otene cleavage has been quite controversial
until recently because both symmetric and
asymmetric cleavage of p-carotene has been
reported in crude extracts, and at least in
some systems, direct formation of retinoic acid
from p-carotene has been observed (4). How-
ever, recent breakthroughs in this field have
throughs can be traced to the first identi-
fication of a maize cDNA encoding 9-cis epoxy-
322 Retinoids

Table 7.2 Human Teratogenicity Associated with Systemic Isotretinoin Therapy

Organ Teratogenicity Observed
Central nervous system Cerebral abnormalities
Cerebellar malformation
Hydrocephalus
Microcephaly
Cranial nerve deficits
Skull Developmental abnormalities
External ear Anotia
Micropinna
Small or absent external auditory canals
Eye Microphthalmia
Heart and cardiovascular Developmental abnormalities
Thymus Developmental failure
Parathyroid gland Functional deficits leading to parathyroid hormone deficiency

carotenoid dioxygenase, the enzyme responsi-
ble for the production of abscisic acid, a carot-
enoid cleavage product that functions as an
important plant growth regulator (5,6). Vogt
and collaborators used cDNA encoding maize
9-cis epoxycarotenoid dioxygenase as a probe
to isolate a metazoan (Drosophila) homolog of
this enzyme and showed that the enzyme,
p-carotene-15,15'-dioxygenase,symmetri-
cally cleaved p-carotene into two molecules of
retinaldehyde (7). Interestingly, mutations in
the Drosophila gene, ninaB, encoding this en-
zyme, cause blindness, because this species
(S), like humans, is dependent on retinoid
chromophores for rhodopsin-based visual sig-
nal transduction (see below). The phenotype
associated with this mutant can be rescued by
dietary administration of retinal, the direct
precursor of the Drosophila visual chro-
mophore, 3-hydroxy-retinal (81, thus bypass-
ing the need for dietary sources of carotinoids.
Hunziker's group then isolated a cDNA encod-
ing the chicken P-carotene-15,15'-dioxygen-
ase (9),and the groups of Blaner and Cunning-
ham isolated the mouse homolog (10, 11)and
demonstrated that this enzyme catalyzed the
symmetrical cleavage of p-carotene into two
molecules of retinaldehyde.
3.3 Asymmetrical Cleavage of @Carotene
Asymmetric cleavage p-carotene has been re-
ported by several groups, and this controversy
also seems to be solved as a result of the clon-
ing of a cDNA encoding an enzyme that asym-

p-carotene

""'4 p-carotene -1 5,l.Y-dioxygenase

Figure 7.4. Symmetrical cleavage of p-carotene.

3 Retinoid Biosynthesis
Carotene dioxygenase
1 Unknown enzymes
Figure 7.5. Asymmetical cleavage o f p-carotene.
metrically cleaves p-carotene to yield two mol-
ecules of p-apocarotenal with different chain
lengths (12) (Fig. 7.5). These p-apocarotenal
cleavage products have been proposed to serve
as precursors to retinoic acid in a retinal-inde-
pendent pathway (13). In this proposed path-
way, it has been suggested that the p-apocar-
otenal cleavage products are subjected to a
series of chain-shortening reactions similar to
/3 oxidation of fatty acids and then oxidatively
metabolized to one molecule of retinoic acid
(13);however, this has yet to be demonstrated
directly. Nonetheless, it is now clear that at
least two pathways of p-carotene metabolism
are possible in humans and other mammals,
whereas Drosophila use only the symmetrical
cleavage pathway. The enzymes responsible
for these cleavage reactions are present in in-
testinal mucosal cells that likely serves as an
important site of p-carotene processing in hu-
mans. The enzymes are also present in several
other tissues and seem to be expressed during
the early stages of embryogenesis, suggesting
that the localized production of retinoids dur-
ing developmental processes is critical.
323
p-ionone (Cll,Cl3, or CIS)
Potentially mechanistically similar to
poxidation of fatty acids
3.4 Formation of Retinyl Esters
Retinaldehyde, when bound to retinol binding
protein I1 (CRBPII), serves as a substrate for .
retinal reductase resulting in the production
of retinol (14), which then binds to cellular
retinol binding protein (CRBP) forming holo-
CRBP. Holo-CRBP seems to be the preferred
substrate for an esterification reaction (Fig.
7.6) mediated by lecithinxetinol acyl trans-
ferase (LRAT), a microsomal enzyme that
uses acyl groups donated from phosphatidyl-
choline (14).In cells not expressing CRBP, ret-
in01 esterification is carried out by a different
enzyme, acyl CoAretinol acyl transferase
(ARAT).
Dietary retinyl esters are also processed in
mucosal cells of the intestine through sequen-
tial de-esterificationlre-esterificationreactions
(14). Retinyl esters are then incorporated into
chylomicrons and pass from the intestine into
the lymph fluid where lipolysis occurs, result-
ing in the formation of chylomicron remnants
that continue to harbor retinyl esters. Chylo-
micron remnants containing retinyl esters en-

trans-retinol
Retinyl ester hydrolases
Figure 7.6. Reduction o f retinal
t o retinol in gut - a n d formation o f trans-retinyl esters
r e t i n y l esters.
ter the general circulation and are taken up
primarily by the parenchymal cells of the liver
and de-esterified to yield retinol. Cellular con-
centrations of free retinol in liver are quite low
because most retinol binds immediately to
CRBP or retinol binding protein (RBP), the
latter of which is a serum transport protein
belonging to a large family of fatty acid bind-
ing proteins. RBP-retinol is secreted by the
parenchymal cells and taken up by the stellate
cells of the liver or target tissues (15, 16).
Within stellate cells, retinol is re-esterified
and stored in lipid droplets in which trans-
retinyl esters account for approximately 42%
of total lipid present (15,16).Palmitate retinyl
esters comprise the majority retinyl esters
present in lipid droplets with lower amounts
of the corresponding stearate, oleate, and li-
noleate esters. Retinyl esters present in the
liver stellate cells account for approximately
75% of total retinoid body stores and are ex-
tremely long-lived (17).
3.5 Mobilization of Body Vitamin A Stores
As extrahepatic demand dictates, retinyl es-
ters stored in the stellate cells of the liver are
mobilized (18).This process involves de-ester-
ification by the action several retinyl ester hy-
drolases, rebinding to RBP, and transport to
Retinoids
NADPH
Short-chain dehydrogenases/reductases
NADP+
CH20H
I
Lecithin:retinol acyl transferase (LRAT)
Phosphatidylcholine
1
the target tissue(s). RBP seems to be critical
for retinol transport to tissues, especially
when the diet is deficient in vitamin A. Mice
that are null for RBP expression exhibit re-
duced levels of serum retinol levels: however.
these mice seem to function normally, at least
with respect to vision, if retinol is supple-
mented in the diet (19). Retinol dissociates
from RBP on reaching these extrahepatic tis-
sues and likely enters the target cell by passive
diffusion (18). Although a receptor-mediated
form retinol uptake has been proposed (20,
. , it is unclear how this contributes to the
21).
overall cellular uptake of retinol which has
been repeatedly shown to enter the cell rapidly
by passive diffusion (18).
3.6 The Fate of lntracellular Retinol
The fate of intracellular retinol is dependent
on cell type. In the eye, trans-retinol can be
esterified by LRAT or isomerized to ll-cis-ret-
in01 by a cell-specific isomerase (22). ll-cis-
retinol is then oxidized to 11-cis-retinal by 11-
cis-retinol dehydrogenase (22). 11-cis-retinal
binds to opsin by forming a Schiff base with an
opsin lysine residue and the resulting complex
is known as rhodopsin (22). Rhodopsin is a
member of the seven transmembrane family
of proteins that serves as receptor for photons
3 Retinoid Biosynthesis
CH20H
trans-retinol
I NAD+or NADPf
Short-chain dehydrogenasesl
reductases Alcohol dehydrogenases
NADH or NADPH
(23). Light-induced isomerization of ll-cis-
retinal plays a central role in the conversion of
visual signals, i.e., light, into nerve impulses
known as the visual cascade (see below) (23).
3.7 Oxidation of Retinoi
In many other cell types, trans-retinol binds to
CRBP and this holo-CRBP serves as the sub-
strate for an oxidative reaction in which the
primary alcohol is converted into an aldehyde
(Fig. 7.7), yielding retinaldehyde (trans-reti-
nd)((18). CRBP plays an extremely important
role both in protecting retinol from non-spe-
cifk oxidative reactions and in the oxidative
reaction to retinaldehyde (18).Indeed, CRBP
physically interacts with the enzyrne(s) re-
sponsible for this oxidation reaction, retinol
dehydrogenase (RDH). Many enzymes, in-
cluding class I, 11, and IV cytosolic alcohol de-
hydrogenases, as well as microsomal RDHs,
short-chain dehydrogenases, and cytochrome
P450 isoforms, have been described that
convert unbound retinol into retinal in a nic-
otinamide adenine dinucleotide phosphate
(NADPt) - or nicotinamide adenine diphos-
phate (NAD+)-dependent manner (14, 24).
With a bewildering number of enzymes capa-
ble of oxidizing retinol to retinal, this area re-
mains a bit of a controversial topic in retinoid
biology (14), but it is currently felt that the
enzymes using holo-CRBP as a substrate (as
opposed to free retinol) may be the most phys-
iologically relevant of the large family of "ret-
in01 dehydrogenases." However, the crystal
structure of CRBP bound by retinol has been
solved at 2.1A, revealing that the protein is
composed of 10 anti-parallel P strands that
Figure 7.7. Oxidation o f r e t i n o l t o r e t i n a l in
cells.
fold into an orthogonal barrel (25). Within the
barrel, trans-retinol was found to exist in a
planar conformation with the free hydroxyl
bonded to Gln108. From these definitive, crys-
tallographic studies, it is not clear how the
hydroxyl group, which is sequestered deep
within the protein, would be available for oxi-
dation in a reaction catalyzed by a retinol de-
hydrogenase as proposed by Napoli (14).
Clearly, the role of holo-CRBPI in the oxida-
tion reaction needs to be clarified, and this will
likely require co-crystallization of halo-CREW
and the retinol dehydrogenase. Thus, the com-
plete picture is still emerging, and a potential
role for the cytosolic alcohol dehydrogenases,
which seem to use only free retinol as the sub-
strate, in retinol oxidation cannot be ruled out
at this time (24).
3.8 Formation of Retinoic Acid: A Major
Signaling Retinoid
Oxidative conversion of retinal (RAL) to reti-
noic acid (RA; Fig. 7.8) is a much less conten-
tious issue because two types of retinaldehyde
dehydrogenases have been clearly delineated
(14). Once formed, RAL binds to CRBP form-
ing CRBP-retinal, which serves a substrate for
both retinaldehyde dehydrogenase 1 and 2
(RALDH1 and RALDH2, respectively), that
catalyzes the irreversible formation of RA
(14). In addition, both enzymes also produce
RA from free RAL, however, free RAL is essen-
tially undetectable in all tissues excluding the
retina (14). Both RALDH enzymes are ex-
pressed in multiple tissues and during em-
bryogenesis; however, the expression pattern
of RALDH2 seems to be much more restricted,

Figure 7.8. Oxidation of retinal to retinoic
acid in cells.
suggesting a role in the localized formation of
RA during development and in the adult or-
ganism (26-28). Indeed, this hypothesis has
been confirmed by genetic means that demon-
strated the essential role of RALDH2 during
embryogenesis (29). Although the two en-
zymes are highly related on the amino acid
level and bind RAL with a reasonably similar
affinity, two findings strongly suggest that
RALDHl and RALDH2 perform distinct func-
tions in vivo. First, the expression of the two
enzymes differs temporally during embryo-
genesis (26-28) and spatially during both em-
bryogenesis and in adult animals (14).Second,
the two enzymes are differentially regulated
by apo-CRBP, which inhibits RALDHl (IC,,
= 1.4 p M ) but not RALDH2 (14). Third,
RALDHl and W D H 2 may be differentially
regulated by RA, presumably acting through
RARs and RXRs to regulate expression of the
corresponding genes (see below) (14).
A third RALDH (RALDH3) has been iden-
tified in the developing chick retina (30, 31).
This enzyme is capable of producing RA from
RAL and appears to be the avian ortholog of
human aldehyde dehydrogenase 6 (30,311. In
addition to the developing retina (which also
expresses RALDHl), RALDH3expression was
also observed in the nasal region (30,311, sug-
gesting that this RALDH may be involved in
both retinal and olfactory system develop-
ment. The relative contributions of each
RALDH enzyme to the overall production of
RA likely depends on both cell type and devel-
opmental stage of the organism. Each of the
RALDH enzymes display a high degree of in-
ter-species conservation, suggesting a com-
Retinoids
CHO
trans-retinal
Retinaldehyde dehydrogenases
1 and 2
NADH or NADPH
trans-retinoic
acid
monality of function and highlighting the im-
portant role of each. Furthermore, Napoli's
group recently reported the isolation of an-
other aldehyde dehydrogenase, ALDH12, ca-
pable of converting retinaldehydes into RA
(see below).
With the exception of the eye and possibly
B and T lyrnphoctes (see below), trans-RA and
its isomers are generally considered to be the
most important retinoids with regard to cellu-
lar signaling. The retinoic acids bind directly
to and activate the numerous members of the
retinoic acid (RAR) and retinoid X (RXR) farn-
ilies of nuclear receptors, all of which function
as ligand-dependent transcription factors. In
turn, these receptors convey the retinoid sig-
nal by regulating the expression of literally
hundreds of RA target genes encoding pro-
teins that are involved in nearly all aspects of
organismal function during development and
in adult life. Indeed, all of the retinoid com-
pounds approved by the FDA for clinical use
activate one or both families of retinoid recep-
tors. The RARs, RXRs, retinoid signaling
mechanisms, and target genes are discussed in
greater detail in following sections.
3.9 Active Isomers of RA
Some isomers of trans-RA have been reported
to exert very potent biological effects. Most
noteworthy of these is 9-cis-RA (see Fig. 7.21,
which was originally demonstrated to be a ret-
inoid that activates both RARs and RXRs,
whereas trans-RA activates only RAR family
members (32-34). Levin and colleagues origi-
nally isolated [3H]9-cis-RA using RXRs to
"trap" it in cells fed [3H]trans-RA, implying
3 Retinoid Biosynthesis
Docosahexaenoic acid
Phytanic Acid
that the cells were capable of the isomeriza-
tion step (32). Heyman and collaborators took
a related approach to arrive at the same con-
clusion independently (35). However, the pu-
tative trans-RA isomerase has not been iso-
lated and isomerization can occur non-
enzymatically (36, 37). In addition, cisoid
derivatives of p-carotene exist that may give
rise to 9-cis-retinol,9-cis-RAL,and ultimately,
9-cis-RA (38). Napoli and Lin recently isolated
a novel retinal dehydrogenase activity corre-
sponding to the previously known human
aldehyde dehydrogenase, ALDH12 (39).
ALDH12 was demonstrated to have a distinct
preference for 9-cis-RAL relative to trans-
RAL, suggesting that this enzyme may be im-
portant for the in vivo formation of 9-cis-RA
(39). Moreover, ALDH12 expression was ob-
served in fetal and adult tissues (liver and kid-
ney) that contain relatively high levels of cis
retinoid precursors such as 9-cis-retinol.
Thus, the possible isomerization of trans-RA
into the 9-cis-RA in vivo does not seem to be
required for the generation of the latter com-
pound.
Other groups have demonstrated that sev-
eral chemical moieties may activate RXR farn-
ily members leading investigators to speculate
that 9-cis-RA may not be the only physiologi-
cal ligand capable of activating RXRs in vivo
(40, 41). Interestingly, neither of the other
RXR-active compounds, phytanic acid and do-
cosahexaenoic acid, is a retinoid, although
both harbor structural motifs similar to natu-
rally occurring retinoids (Fig. 7.9).
An unsaturated derivative of retinoic acid,
3,4-didehydroretinoic acid (Fig. 7.101, has
been demonstrated by Eichele and Thaller to
be an important signaling molecule in a model
system of limb development, the chick limb
bud assay (42). 3,4-Didehydroretinoic acid is
derived from 3,4-didehydroretinol, which is
Figure 7.9. Structures of alternative RXR
agonists.
also known as vitamin A2. However, with the
exception of cultured human keratinocytes ex-
posed to high concentrations of retinol (431,
this form of retinoic acid has not been detected
in mammalian tissues to any appreciable de-
gree (14). Thus, the potential role of 3,4-dide-
hydroretinoic acid in the vitamin A signaling
pathways in mammals is not clear at the
present time.
Hiimmerling and his colleagues have identi-
fied a novel pathway of vitamin A action in
lymphocytes that does not involve RA.These
investigators originally discovered that retinol
was required for B-cell proliferation (44) and
T-cell activation (45) and that RAL, but not
RA, would substitute for retinol in these ac-
tions. These investigators have subsequently
discovered that 14-hydroxy-4,14-retro-retinol -
and 13,14-dihydroxyretinol(Fig. 7.11) bind to
and augment the activation of both cRaf and
protein kinase Ca by reactive oxygen (46,471.
cRaf and protein kinase Ca are serine-lthreo-
-
nine-kinases that play important roles in sig-
nal transduction cascades, such as that associ-
ated with activation of mitogen-activated
protein (MAP) kinase. If these data are con-
firmed, both cRaf and protein kinase Ca can
also be considered as receptors for retro-reti-
noids. Hammerling et al. has proposed that
retro-retinoids may function to facilitate elec-
tron transfer allowing the efficient redox acti-
3,4-didehydro-trans-retinoic acid
Figure 7.10. 3,4-didehydroretinoicacid.

Anhydroretinol
Figure 7.11. Retro-retinoids.
vation of both kinases (47). These investiga-
tors have also isolated another compound,
anhydroretinol (Fig. 7.11), that functions as
an antagonist of 14-hydroxy-4,14-retro-reti- CRABPs remains an open question.
no1 action in the lymphocyte context (48). An-
hydroretinol not only antagonizes the effects
of 14-hydroxy-$14-retro-retinol on B-cell pro-
liferation and T-cell activation, but itself rap- Tissue-specific catabolism of RA is extremely
idly induces T cell death (48). Thus, it seems
that 14-hydroxy-4,14-retro-retinol and anhy-
droretinol, both of which are produced by lym-
phocytes from retinol, act back on these cells
to regulate homeostasis in opposing direc-
tions.
Unlike many retinol dehydrogenases, the
enzyme that catalyzes the conversion of reti-
no1 to anhydroretinol, retinol dehydratase,
does not use CRBP-retinol as a substrate;
rather, it uses free retinol (49-51). However,
the enzyme responsible for biosynthesis of 14-
hydroxy-4,14-retro-retinolhas not yet been
isolated.
3.1 1 Non-Receptor, RA Binding Proteins
Retinoic acids, particularly trans-RA, bind
with high affinity to cellular retinoic acid bind-
ing proteins I and I1 (CRAEiP I and C W P 11).
Like the CRBPs, CRAEiPs are widely ex-
Retinoids
pressed in the embryo and adult and are found
in essentially all RA target tissues. Three
functions have been ascribed to the CRABPs:
(1)it is generally believed that the C W P s
serve to buffer intracellular concentration of
RA by binding free RA, (2)CRABP may facil-
itate metabolism of RA in that CRABP-RA
complexes are better substrates for oxidative
enzymes than is free RA (14), and (3) CRABPs
may also function to transport RA to the nu-
cleus, the loci of action of the nuclear receptors
RARs and RXRs (18).Functions 1 and 2 would
serve to reduce the cell's sensitivity to RA, and
this is consistent with overexpression studies
in F9 embryonal carcinoma cells (52). How-
ever, both C W P I- (53, 54) and II- (55, 56)
null animals are remarkably normal, as are
the double knock-out (CRABP I-'-/CRABP
II-/-) animals (55). Moreover, none of these
animals are more sensitive to RA-induced tox-
icities than animals expressing wild-type
CRABP I and I1 (55). If CRABP I or I1 play a
role in "buffering" intracellular concentra-
tions of RA or in the delivery of RA to the
nucleus as hypothesized, it is unclear why
knock-out animals do not display more of a
phenotype. Thus, the biological role(s) of the
4 RETINOID METABOLISM
important in terminating the retinoid signal,
and ultimately, in dictating the tissue respon-
siveness to RA. Thus, the metabolism of RA
and related compounds has generated a large
amount of interest for both clinicians and ba-
sic scientists. Consistent with other aspects of
retinoid biology, metabolism of RA and RA
precursors is a complex area. First, one of the
major pathways of RA metabolism is gluc-
uronidation at the carboxyl group (Fig. 7.12).
This reaction is catalyzed by the liver enzyme
UDP-glucuronyl transferase and the product
of the reaction, retinoyl-p-glucuronide, partic-
ipates in a large degree of enterohepatic circu-
lation. Napoli has suggested that retinoyl-B
glucuronide may serve as a water-soluble,
reclaimable pool of RA to prevent retinoid de-
ficiency (14).
Second, oxidative metabolism (hydroxyla-
tion) of RA occurs at both the C4- and C18-

positions of the p-ionone ring yielding 4- and
18-hydroxy RA, respectively, and this hy-
droxylation has been shown to be induced by
RA (57, 58). The relative importance of the
two pathways of oxidative metabolism are un-
known, but oxidation at the 4-position has
been studied more extensively. 4-Hydroxy RA
is further metabolized to give 4-0x0 RA that
then undergoes isomerization to yield 4-0x0-
13-cis-RA (59-61). Together, the retinoyl-p-
glucuronide and the C4-oxidation products
account for most of the steady-state metabo-
lites of RA (59-61). Minor metabolic prod-
ucts include the 5,6-epoxy-trans-RA (61)
(see Fig. 7.12).
C4-oxidation of RA is catalyzed by cyto-
chrome P450 as suggested by the auto-induc-
tion of RA metabolism at this position (see
above) and the inhibition of the hydroxylation
reaction by ketoconazole and related com-
pounds that are non-specific inhibitors of
many members of the cytochrome P450 family
(62). Indeed, Petkovich and colleagues re-
cently identified a cytochronie P450 family
member that hydroxylates RA at both the C4-
and C18-positions in a NADPH-dependent
manner, and furthermore, oxidizes 4-hydroxy
RA to 60x0 RA (63). The expression of this
enzyme, Cyp26A1, is induced by RA in several
cell lines and tissues, and animals lacking the
enzyme display many of the features of hyper-
vitaminosis A, particularly teratogenesis (64),
suggesting that Cyp26A1 plays an important
role in RA metabolism in the developing em-
bryo. Recently, a second member of the Cyp26
family was described, Cyp26B1 (65-67), that
displays a pattern of expression different from
that of Cyp26A1, suggesting that each enzyme
may play a distinct role in RA metabolism.
Both enzymes exhibit similar substrate speci-
ficity and kinetic parameters (66). Interest-
ingly, the 4-0x0 metabolites of RA are better
substrates for UDP-glucuronyl transferase-
mediated glucuronidation than trans-RA (681,
suggesting that hydroxylation of RA at the C4-
position may facilitate conjugation and elimi-
nation.
The induction of Cyp26A1 expression by
RA is of great clinical relevance because in-
creased expression of the enzyme potentially
reduces the intracellular pool of endogenous
or pharmacologically administered trans-RA
Retinoids
that is available to activate the retinoic acid
receptor subtypes. Because the receptors likely
mediate the beneficial effects of trans-RA in a
therapeutic sense, this renders trans-RA
treatment less efficacious in the application
for which it is being used. This may be a par-
ticular concern for the use of trans-RA in some
types of breast cancer as Petkovich and col-
leagues have shown that Cyp26Al is strongly
up-regulated by both trans-RA and 9-cis-RAin
MCF-7 breast cancer cells (69).The Petkovich
group and others have also shown that
trans-RA induces Cyp26A1 expression and its
own metabolism in many other cell types in-
cluding human leukemic cells and cervical
cancer cells, suggesting that the problem of
auto-induction of trans-RA metabolism may
generalize to other types of cancer cells. Clin-
ically, it may be possible to circumvent this
problem using at least two strategies: (1)use
of retinoid analogs that are not Cyp26Al or
Cyp26B1 substrates or (2) concomitant use of
trans-RA and an inhibitor of Cyp26A1 andlor
Cyp26B1 that would result in prolongation of
the half-life of trans-RA in the cancer cell. The
synthesis of specific Cyp26Al/Cyp26Bl inhib-
itors capable of augmenting RA action in can-
cer cells represents a potentially exciting new
area of drug development.
5 ROLE OF RETINOIDS IN VISUAL
SIGNAL TRANSDUCTION
Metabolites of retinol play a crucial role as
visual pigments in the visual systems of all
vertebrate and invertebrate animals. Humans
and higher vertebrates use 11-cis-retinal as
the primary visual pigment (221, whereas
other species use 3,4-didehyrdoretinal (fish,
amphibians, and some reptiles), (3R)and (3s)-
3-hydroxyretinal (insects), and (4R)-4-hy-
droxyretinal (cephalopods). In all cases, the
role of the retinoid metabolite is similar: pho-
tons induce isomerization of a visual pigment
which then initiates a cascade of nervous im-
pulses that are transmitted to the regions of
the brain that process visual signals (22, 23).
The role of 11-cis-retinal in the mammalian
visual cycle is very well understood at the mo-
lecular level. This cycle is composed of three
components: (1)light detection, (2) initiation
5 Role of Retinoids in Visual Signal Transduction
of signal transduction, and (3) regeneration of
rhodopsin, which functions as a "receptor" for
5.1 Photon Detection
Detection of photons in the mammalian visual
system is accomplished by the light-induced
isomerization of 11-cis-retinal to all-trans-ret-
i d . The process is similar in both rods, which
serve essentially as light detectors, and cones,
ich are capable of color recognition (22).
wever, the detection kinetics, regeneration
me, and signal and wavelength sensitivities
between the two types of vision-sensing
ptor" in all cases is rho-
ts of a protein (opsin) in
complex with 11-cis-retinal through the for-
mation of a Schiff base (23). The light-induced
isomerization of 11-cis-retinal induces a con-
formational change in the associated protein
nderlies activation of the
5.2 Initiation of a Visual Signal
Rhodopsin is a member of the seven trans-
membrane family of guanine nucleotide bind-
led receptors (70, 71). This
very large number of pro-
ceptors for neurotransmit-
rants, and gustants (com-
pounds that generate a taste sensation) (70,
71). All of these receptors signal through gua-
nine nucleotide binding (G)-proteins. How-
ever, rhodopsin is unique in two respects.
First, rhodopsin has a bipartite structure com-
posed of a protein component (opsin) and 11-
cis-retinal in a protonated Schiff base linkage
with the €-amino group of l y ~ i n e ~(bovine
'~
opsin) (22, 23, 70, 71). Second, in contrast to
other G-protein-coupled receptors, the "ago-
hodopsin is a photon of
chemical, peptide, or hor-
e (71). As described above, a single photon
ght induces the isomerization of ll-cis-
tinal to all-trans retinal (Fig. 7.13). The
n associated with light-in-
ced isomerization of 11-cis-retinal is trans-
ced through the Schiff base to the opsin
in, resulting in the for-
conformation of rhodop-
(metarhodopsin11,hereafter referred to as
R*; see Fig. 7.13). R* interacts with another
membrane protein, transducin (Gt), the
G-protein of this signaling cascade. Gt, like
other G-proteins, is a heterotrimeric complex
composed of a, P, and y subunits. In the inac-
tive state, the a subunit of transducin (Gt,) is
bound by GDP. However, interaction with ac-
tivated R* promotes rapid GTP-GDP ex-
change on Gt, with concomitant dissociation
of holo-Gt into Gt, (that is now bound by
GTP) and GtPysubunits. Although this dis-
cussion focuses on the role of Gt,-GTP, an im-
portant role for GtPYis not ruled out (71). Gt,-
GTP is known to be an important activator of
a membrane-associated, cGMP phosphodies-
terase (PDE), and this occurs through the di-
rect interaction of Gt,-GTP with PDE and
subsequent displacement an inhibitory sub-
unit of PDE (22,23, 70, 71). In the dark state,
high intracellular levels of cGMP activate a
cGMP-gated cation channel that conducts pri-
marily Nat and Ca+ (22,23, 70, 71). The cur-
rent through this channel, which is known as
the "dark current," determines the resting po-
tential of the dark-adapted rod. However, ac-
tivated PDE rapidly cleaves cGMP, lowering
intracellular levels of cGMP and resulting in
the shutting off the dark current (22). This
results in a rapid hyperpolarization of the rod.
On one side, the rod cell detects photons
throughout the above-described mechanism.
However, the opposing side of the rod is in
synaptic contact with other retinal cells such
as both horizontal and bipolar cells and these
synapses use glutamate, an excitatory neuro-
transmitter (22, 23, 70, 71). Hyperpolariza-
tion of the rod reduces its excitability, which
results in a decrease in the amount of gluta-
mate released by the cell onto horizontal and
bipolar cells (22). This signal is then trans-
duced through the layers of the retina and ul-
timately to the optic nerve that projects to,
among other areas, the visual cortex. The
same signal also inhibits signaling by neigh-
boring cells and this "surround inhibition"
provides a mechanism for the precise localiza-
tion of stirnulatory input, i.e., light, into the
visual system (22, 70, 71).
5.3 Shutting Off R*
The a subunit of transducin, like other G-pro-
teins, harbors an intrinsic GTPase activity

Photon
Gtafly -
Figure 7.13. Visual system signaling and the
role of 11-cis-retinal.
that hydrolyzes bound GTP to GDP, thereby
terminating signaling by Gt,-GTP (71). The
Gt, GTPase activity is greatly stimulated by a
GTPase accelerating protein (GAP) complex
composed of the regulator of G-protein signal-
ing (RGS) 9 protein and a G-protein P subunit
(Gp5) (22). The GAP complex rapidly cata-
lyzes inactivation of Gt,-GTP to Gt,-GDP, the
latter of which does not interact with or acti-
vate PDE. Thus, the inhibitory and catalytic
subunits of PDE rebind with a concomitant
decrease in intracellular PDE activity and a
rise in cGMP levels that results in reactivation
of the dark current (23).
An interesting form of self-regulation also
occurs at the level of activated rhodopsin. R* is
the substrate for a kinase known as rhodopsin
kinase that phosphorylates the protein at mul-
tiple serine and threonine residues in the car-
boxyl terminus of the protein (70). Although
this phosphorylation somewhat compromises
the ability of R* to interact with and activate
transducin, complete inactivation of R* re-
quires both rhodopsin kinase-mediated phos-
phorylation of R* and the binding of another
protein, arrestin, to phosphorylated rhodop-
sin (71). The phosphorylated rhodopsin is sub-
sequently dephosphorylated by a protein
phosphatase 2A isoform to generate native op-
Retinoids
Rhodopsin
1 1-&retinal
(R)
-1
Rhodopsin
trans-retinal
(R*)
I
GDP/-- Gbpy GTP
GDP
Gta - GTP + Gtpy
GTP
PDE
1
- PDE*
cGMP
1
- GMP
1
K+ Channel
(Dark current)
sin, which can then recombine with 11-cis-ret-
inal to reform rhodopsin (see below) (22).
5.4 The Retinoid Cycle in Visual Processes
The retinal pool of 11-cis-retinal is clearly crit-
ical for photon detection and visual signal
transduction. There are two key aspects re-
garding this that are important to consider:
the precursor(s) from which 11-cis-retinal de-
rived, and, bioconversion of isomerized trans-
retinal back to 11-cis-retinal that can recom-
bine with opsin to re-form rhodopsin. The
latter aspect is obligatory for restoration of the
dark state, regenerating a photosensitive re-
ceptor capable of undergoing another cycle of
photon detection and signal transduction.
Considered together, these events represent
the retinoid cycle in the visual process (22).
The entire process of re-isomerization and for-
mation of a new rhodo~sinmolecule occurs in
two different retinal tissues and involves sev-
eral enzymatic steps that are described below
(see Fig. 7.14).
Dietary sources of vitamin A also provide
the metabolites that are necessary for vision.
The ingestion and processing of p-carotene or
retinyl esters to retinol and the transport of
RBP-retinol complexes to target tissues was
discussed earlier in this chapter, and the ret-
 de of Retinoids in Visual Signal Transdudion
RhodoPsin 11-&-retinal
11-cis-retinal
Visual
system
signal
transduction
Figure 7.14. Retinoid cycle in vision.
ina should be simply viewed as a target tissue
at l;his level. However, the retinal production
of 11-cis-retinal from retinol and retinol es-
ter;s, which is reasonably specific to the eye, is
described below.
As described above, photon detection at the
lev1el of rhodopsin occurs in the rod (black and
white vision) or cone (color vision) outer seg-
ments. Thus, this is the locus of the first
ste:p of the so-called "retinoid cycle." After
~ h~toisomerization,
( all-trans-retinal remains
bo1ind to R* but eventually diffuses out of the
ret.inal binding pocket of opsin in a process
tha~tis not completely understood (22). Pas-
siv~2 diffusion is probably the most important
det erminant of trans-retinal dissociation from
the protein, but the existence of an ATP-de-
Per)dent transporter that may facilitate this
Process has been demonstrated in some sys-
ten1s (22).Once dissociated from opsin, trans-
ret:inal is reversibly reduced to all-trans reti-
no1 by a member of the membrane-associated
shc~rt-chainalcohol deydrogenase family that
is 1mown as all-trans-retinol dehydrogenase
(RIIH). The all-trans-retinal + all-trans-reti-
no1conversion occurs in the outer segments of
both rods and cones, is dependent on NADPH,
ant1 may be the rate limiting step in the regen-
eration of 11-cis-retinal (22). Several forms of
RDH capable of reducing all-trans retinal to
333
retinol exist in the retina and the enzymes
responsible for this reaction in rods and cones
seem to be distinct.
All-trans retinol then diffuses out of the
outer segments (rod or cone) and into the ret-
inal pigmented epithelium (RPE). The diffu-
sion of all-trans-retinol out of the outer seg-
ments and into RPE cells is facilitated by
interstitial retinoid binding protein (IRBP),
which is localized in the extracellular matrix
of the retina (22). IRBP binds all-trans-retinol
as it diffuses out of the outer segment cells and
is believed to facilitate transport of all-trans-
retinol to the RPE cell. Additionally, RPE cells
directly acquire retinol from serum as de-
scribed earlier. In both cases, retinol in the
RPE cell is rapidly esterified by LRAT in a
lecithin-dependent mechanism (22). Retinyl
esters provide both a mechanism of storage for
the RPE cell and/or provide a substrate for the
isomerization reaction (22).
The isomerization reaction to ll-cis-reti-
no1 likely occurs through one of two possible
mechanisms. First, the all-trans-retinyl ester
may be a substrate for an as yet identified
"isomerohydrolase" that uses the free energy
of retinyl ester hydrolysis to drive the isomer-
ization reaction (22). In this case, the product
of the reaction, 11-cis-retinol,would be gener-
ated in essentially one step by a single enzyme,

the putative isomerohydrolase, which has not
been isolated. Second, the two reactions may
be distinct, i.e., retinyl ester hydrolases in
RPE cells may hydrolyze the all-trans-retinyl
esters generating all-trans-retinol that is sub-
sequently isomerized to 11-cis-retinolthrough
a carbocation intermediate. Although the lat-
ter reaction is endothermically unfavorable
(AG = +4 kcal/mol), McBee and colleagues
have proposed that retinol binding proteins,
such as CRBP, may drive the reaction forward
(72). 11-cis-Retinol,formed by either or both
of the above pathways, is then oxidized to 11-
cis-retinal by one or more 11-cis-retinoldehy-
drogenase, which are members of the short-
chain alcohol dehydrogenase family (22, 71).
This reaction is well understood at the molec-
ular level, and mutations in the RDH5 gene,
which encodes an 11-cis-retinol dehydroge-
nase, is associated with fundus albipunctatus,
a human disease in which there is delayed
dark adaptation in both cones and rods be-
cause of the impaired regeneration of ll-cis-
retinal (73-78). Cellular retinaldehyde bind-
ingprotein (CRALBP) has been proposed to be
important in both the isomerization (all-
trans-retinol + 11-cis-retinol)and the oxida-
tion (11-cis-retinol+11-cis-retinal) reactions
(22); however, the underlying molecular
mechanisms have not been identified. None-
theless, mutations in the CRALBP gene are
associated with compromised retinal disease
such as retinitis punctata albescens (79-821,
suggesting that CRALBP may play a role in
the retinoid cycle in the retina. Additionally,
CRBP I is expressed in RPE and has been sug-
gested to play a role in chaperoning retinol
within the cell and, like CRALBP, possibly has
a role in the isomerization andlor oxidation
reactions (22). Mice devoid of CRBPI have
been generated and exhibit a large reduction
in retinyl ester accumulation in liver (83);
however, it is unknown if these mice have de-
fects in the retinal retinoid cycle.
Finally, an abundant protein in the RPE,
RPE65, seems to play a role in the retinoid
cycle, but the exact role remains a mystery.
Mice lacking this protein accumulate all-
trans-retinyl esters that concentrate within
lipid droplets in RPE cells (84). These mice are
completely devoid of 11-cis-retinal and other
cis-retinoid metabolites and are severely com-
Retinoids
promised in visual processes. However, the de-
fective vision phenotype of these animals is
reversed by dietary administration of 9-cis-
retinal-which can also form a Schiff base
with rhodopsin (creating isorhodopsin) that
functions as a photon detector in the visual
process similar to 11-cis-retinal (84). Interest-
ingly, RPE65 shares around 40% sequence
identity with the dioxygenase that catalyzes
the symmetrical cleavage of p-carotene into
two molecules of retinal (7, 8); however, the
functional significance of the relationship to
the p-carotene dioxygenase is completely un-
known.
11-cis-retinal then diffuses out of the RPE
cell and back into the outer segment photore-
ceptor cell. It is presently unknown if the
movement of 11-cis-retinal to the photorecep-
tor cell requires interaction with IRBP, but
this is clearly a possibility. Once in the photo-
receptor cell, 11-cis-retinal reacts with opsin
through formation of a Schiff base as de-
scribed above (Fig. 7.14).
5.5 Retinoids and the Basis of Color Vision
The absorption maxima of free 11-cis-retinal
is in the near-ultraviolet range (360-380 nm).
However, the absorption maxima of the 11-
cis-retinylidene moiety in the context of a pro-
tonated Schiff base linkage with rod opsins is
shifted to approximately 500 nm, squarely in
the middle of the visual range. Two factors
contribute to this dramatic red shift in the ab-
sorption maxima of 11-cis-retinylidenegroup
relative to that of free 11-cis-retinal: (1)pro-
tonation of the Schiff base and (2) the exis-
tence of intramolecular negative charges con-
tributed by the opsin component of rhodopsin
(22). One of these negative charges is derived
from Glu113, a residue that is conserved
among all known vertebrate visual pigments.
Glu113is located in the highly hydrophobic en-
vironment of opsin transmembrane domain 2
and provides the counterion for the positively
charged Schiff base (22). The existence of this
counterion in such a hydrophobic environ-
ment shifts the pKa of the protonated Schiff
base by 7 log units, effectively preventing
spontaneous hydrolysis of the ll-cis-retinyli-
dene group.
The recent elucidation of the crystal struc-
ture of rhodopsin bound by 11-cis-retinal at
Retinoic Acid Signaling Pathways
.Igure 7.15. Rhodopsin crystal structure. Three-
imensional structure of rhodopsin based on X-ray
rystallography (186). Note that all-trans-retinal is
rotected from the intradiscal side by multiple
tructural elements, including several P strands.
iarbohydrates are in blue, 11-cis-retinal is in green,
nd helices are in gray. Red shadows are added for
sthetic reasons. This figure was generated by C.
lehnke (Universityof Washington)and is reprinted
iith permission from Prog. Retin. Eye Res., 20,
69-521 (2001).See color insert.
.8A allows direct visualization (Fig. 7.15) of
he spatial arrangements of the transmem-
rane domains of rhodopsin and the location
f 11-cis-retinal in the pocket (85). These in-
estigators found that 11-cis-retinal contrib-
~testo the inactive conformation of rhodopsin
iy holding the transmembrane regions of the
lrotein together. Although the structure of
ileached rhodopsin, i.e., that containing 11-
runs-retinal, was not reported, one can easily
mage the magnitude of the rhodopsin confor-
national change that must accompany light-
nduced isomerization of the chrornophore.
1-cis-retinal.
The above-described structural studies
onfirmed that interactions of the ll-cis-reti-
nylidene group with a cluster of amino acids of
the opsin component dictate the absorption
maxima of rod-derived rhodopsin and also pro-
vide a basis for the visual perception of color in
cone cells of the retina. Although the chro-
mophore is attached to cone opsins through an
identical, protonated Schiff base linkage and
likely occupies a similar position in the opsin
pocket, the absorption maxima of the cone
rhodopsins differ significantly from that of rod
rhodopsin. However, in contrast to those ex-
pressed in monochromatic rod cells, cone vi-
sual pigments have absorption maximas that
cover the visual spectrum. This is believed to
result largely from differential interactions of
11-cis-retinylidenegroup with the side chains
of amino acids lining the cavity in which the
chromophore resides (22, 85). As a result of
these differential interactions, visual pig-
ments present in cones are activated by light
of varying wavelengths giving rise to the per-
ception of color.
6 RETINOIC ACID SIGNALING
PATHWAYS
6.1 Historical Perspective
Naturally occurring retinoic acids and syn-
thetic derivatives exert cellular effects by
binding to and activating two families of nu-
clear receptors (NRs), retinoic acid receptors
( W s ; Fig. 7.16) and retinoid X receptors
(RXRs; Fig. 7.17). Both classes of retinoid re-
ceptors are comprised of three subtypes, a, p,
and y, all of which belong to the steroid and
thyroid hormone receptor superfamily of li-
gand-dependent transcription factors (1).
RARa was the first member of either retinoid
receptor family to be identified simulta-
neously and independently by the groups of
Chambon (86) and Evans (87), both of which
isolated RARa using a low-stringency, cDNA
library screening strategy. Subsequently, the
Chambon (88) and Pfahl (89) groups isolated
RARp and made the realization that the locus
of this gene was a site of viral integration caus-
ing hepatocellular carcinoma. The Chambon
group then cloned cDNA encoding RARy and
demonstrated that this receptor was highly
expressed in skin (90, 91), long known as a
primary retinoic acid target tissue. The Cham-

hRARa H2N
I-A-+B+C----.t D a
hlm (RARa) 98 100 98 98
hlm (RARB) 94 100 100 98
hlm (RARy) 98 100 100 100
Figure 7.16. Retinoic acid receptor subtypes.
bon group also discovered the existence of
multiple isoforms of RARa (92), RARP (931,
and RARy (94),all of which were generated by
alternative splicing and differential promoter
usage (Fig. 7.18). RXR isoforms, particularly
of RXRp (see below), have also been described
(95). Finally, the Chambon group demon-
strated that the retinoic acid receptors and all
of the retinoid binding proteins exhibit spa-
tially and temporally distinct expression pat-
terns in the developing fetus and adult ani-
mals (96-99), suggesting that each may play a
specific role in the retinoic acid signaling path-
ways. This flurry of activity in the time of 3-4
years, driven entirely by molecular biology ex-
pertise of a few groups, completely revitalized
vitamin A research throughout the world. For
the first time, nutritional and metabolic bio-
chemists, toxicologists, and cancer, cell, and
molecular biologists could begin to address the
molecular basis of vitamin A action in health
and disease.
6.2 Discovery of RXRs
As all of the above action was occurring, the
group of Evans continued to isolate cDNAs en-
Retinoids
COOH
E - F--I
99 90
99 92
100 58
coding novel retinoic acid receptor-like pro-
teins and in, the process, identified RXRa
(100). This receptor was so-named bec,ause
trans-RA activated the receptor in tramrient
transfection experiments, however, on1Y at
concentrations much higher than thosc? re-
quired for activation of the M s . This finding
led Mangelsdorf and colleagues to propose
that the ligand for RXRa was a metabolite, the
" X metabolite, of trans-RA (100). This srpec-
ulation was later proven by both the groups of
Levin (32) and Evans (35), both of which iden-
tified 9-cis-RA as the RA "metabolite" respon-
sible for activation of RXR in cultured cells
(see above). However, the parent compolund-
metabolite relationship has never beer1 di-
rectly demonstrated for trans- and 948
and it is possible, as described above, thalt the
two isomers of RA are interconverted in a non-
enzymatic reaction as suggested by the g;roup
of Rando (36, 37). Nonetheless, both the
groups of Levin and Evans made a fundanen-
tally important discovery in identifying a li-
gand, 9-cis-RA, that bound directly to an1d ac-
tivated both families of retinoid receptors,
whereas trans-RA only bound and activ.ated
 6 Retinoic Acid Signaling Pathways

I-A-tB-C+ D a E -
1 55 132 201 259 462

hRXRa H2N COOH

1 124 203 271 330 533

hRXRB HN
, COOH

f
18
+
32
t
90
1 i61i136 205
hRXRy HN
, COOH
i

iE
k
1k I - A - k B t C - D a E -
i hlm (RXRa) 89 96 100 98 99
i
f
hlm (RXRB) 89 99 100 95 99
E hlm (RXRy) 93 100 100 95 99

Figure 7.17. Retinoid X receptor subtypes.

RAR family members. This finding was the

basis for the future development of receptor-
elective retinoids.
.3 Identification of Retinoic Acid
arget Genes
Ilearly, RARs and RXRs bound ligand, and
his was demonstrated directly as described
bove, but these proteins also belong to the
IR superfamily of sequence-specific DNA
inding transcription
- factors. Thus, the next
ajor question that required an answer was
hat is the nature of the DNA response ele-
lent to which the RARs and RXRs bind?"
s was an extremely important question be-
use the answer would provide clues regard-
~g the nature of RA targ& genes. At the time,
Dme response elements for steroid and thy-
3id hormones were known and these were
enerally corresponded to inverted repeats of
hexanucleotide sequence, for example AG-
CT for estrogen receptors or
TTCT for glucocorticoid re-
ptors (where x corresponds to any nucleo-
Ae). In both cases, the hexanucleotide se-
-
quence for each half-site is repeated on the
opposite strand, also in the 5' 3' orienta-
tion. Importantly in the search for retinoic ac-
id-responsive genes, de The and colleagues
had previously identified RARP as a trans-RA-
induced gene in hepatoma cells (101) and
subsequently identified the DNA response
element conferring this inducibility as a
direct repeat (DR) of the sequence GT-
TCACxxxxxGTTCAC (102). This crucial find-
ing was unusual because, although the se-
quence of this retinoic acid response element
( W E ) hexanucleotide was similar to that of
estrogen and glucocorticoid receptors, the rel-
ative orientation of the two half-sites was not.
In contrast to inverted or palindromic repeats
of steroid hormone response elements, the
head to tail orientation of the direct repeats
generates an asymmetrical binding site that is
typical of many non-steroid hormone recep-
tors. Using a different approach, the group of
Evans came to a similar conclusion, defining
what was referred to as the "3-4-5 rule" of
response element recognition in which di-
rectly repeated response elements spaced by 3,
 Retinoids

DBD LBD

mRARal I---
B
AUG A1
mRARa2
-.._

AUG A2 B

Figure 7.18. Schematic organization of

5' region of mouse RAR genes and major
isoforms. There are two major isoforms of
mRARj3l ---
M a and M y , which in both cases,
AUG A1 A3 B
arise from differential usage of two pro-
moters, P1 and P2. RARPl and RARP3 are
transcribed from the P1 promoter, but dif- mRARj33 rn ---
fer as a result of alternative splicing, AUG B
whereas RARP2 and RARP4, which are ---
both transcribed from the downstream P2 AUG A2
promoter, differ in that the P4 isoform
uses a CUG initiation codon and is alter-
natively spliced such that is it is virtually
devoid of an A region. Exons are indicated
by boxes and numbers (El- E5, E8, and
E9). Black and white boxes represent
translated A region sequences and the 5'-
UTR, respectively. For a given RAR type
(a, p, or y), A1-A4 and B represent the
isoform-specific A region and common B
regions, respectively. Reprinted with per-
mission from Trends Biochem. Sci., 17,
AUG A2 B
427-433 (1992).

4, and 5 bp functioned specifically as vitamin
D, thyroid hormone, and RA response ele-
ments, respectively (103). Chambon's group
then extended this rule by demonstrating that
the RA inducibility of the CRBPII gene was
conferred by a directly-repeated RARE spaced
by 2 bp (104), while Mangelsdorf and col-
leagues demonstrated that the complex, di-
rectly repeated response element spaced by
1 bp (DR1) of the rat CRBPII promoter con-
ferred RXR-mediated inducibility, thus identi-
fying the first RXR response element or RXRE
(105). Durand and colleagues then demon-
strated that the mouse CRABP I1 promoter
harbored both DR1 (RXRE) and DR2 (RARE)
elements, indicating that both families of ret-
inoid receptors may be involved in the up-reg-
ulation of this gene by retinoic acids (106).Al-
though numerous groups have shown that
DR1 elements are promiscuous and confer
regulation by several other nuclear receptors,
the molecular basis for response element rec-
ognition and, therefore, target gene regula-
tion, by RARs and RXRs was well-defined by
the above-described body of work. Thus, the
3-4-5 rule was modified to account for DR1
(RXR and many other orphan receptors), and
DR2 (RA)response elements and became com-
monly known as the 1-5 rule (Fig. 7.19). To-
day, many more retinoid-responsive genes
6 Retinoic Acid Signaling Pathways
COUPS 5'
"P
J
"(
6
inoid signals are
he expression of a wide array
plicated in mediating the
eiotropic effects of RAs on cellular function.
de transcription factors
7-113), metabolic enzymes (114), growth
r receptors (115), extracellular matrix
16,117),secreted proteins that are
tulated to convey positional information in
developing embryo (118-120), and genes
oding pro-apoptotic proteins such as
d RXRs Bind Response Elements
a Heterodimeric Complex
era1 groups working independently simul-
eously discovered that RAR family mem-
DEGREES
OUT OF
Figure 7.19. Directly repeated response
elements for members of the retinoid recep-
tor family (RXRand RAR). The sequence of
each half-site is similar, but the spacing be-
tween the two half-sites is distinctive. In-
creasing the length of the intra-repeat
spacer alters the relative orientation of the
two half-sites as indicated on the right side
the figure. For example, the centers of
118
each half-site are on different sides of the
DNA helix in the context of a DR1 response
element (118"out of phase),but only 14"and
21" out of phase in the context of DR4 and
DR5 response elements, respectively.
bers heterodimerize with RXR family mem-
bers and bind all known RAREs as a RAR.RXR
heterodimeric complex (122-126). During the
course of this work, two additional members of
the RXR family, RXRp, which was originally
identified as H-2RBPII by the group of Ozato
(1271, and RXRy, were identified (123, 128),
both of which were also shown to heterodimer-
ize with RAR family members (123). Finally,
the Chambon group used genetic means to
demonstrate that RAReRXR complexes are the
functional units of both retinoid signaling
pathways during mammalian development
(129). Considered together, these remarkable
findings indicate that the two families of reti-
noid receptors, RARs and RXRs, which arose
independently during evolution and harbor

Table 7.3 Comparison of Amino Acid
Sequence Conservation among RAR and
RXR Ligand Binding Domains
RARa RAW
RXRa 28 32 30
RXRP 29 30 30
30 33 30
ERa 23 24 24
- -
Percent identities between the ligand binding domains
of RARs, RXRs, and estrogen receptor (ERa).
little sequence identity outside the DNA bind-
ing domain (see below and Table 7.3), physi-
cally interact in the context of a heterodimeric
complex to regulate the transcription of most
retinoic acid target genes. RXR was subse-
quently shown to heterodimerize with several
other nuclear receptors (Table 7.41, a finding
that implies activators of RXR may potentially
affect a great number of other signaling path-
ways (130,131). Thus, the development of se-
lective, RXR agonists is particularly attractive
to medicinal chemists (see FUTURE PER-
SPECTIVES).
RAR.RXR heterodimeric complexes bind
to a directly repeated, hexanucleotide motif
of the consensus sequence 5'-PuGGTCA-3'
(where Pu represents a purine, see above). Di-
rectly repeated response elements, in contrast
to inverted repeats, are not symmetrical, and
this renders the microenvironment of each
half-site distinct (38). This situation implies
that the orientation of RAR.RXR complexes
RAREs may be ordered and occur in a non-
random manner. Indeed, this is the case: Perl-
mann and colleagues used biochemical tech-
Table 7.4 RXR Heterodimerization Partners
Receptors with Known Ligands
- -
Retinoic acid receptors (RARa, P, y)
Vitamin D receptor (VDR)
Thyroid hormone receptors (TRa, P )
Perooxisome proliferator-activated
receptors (PPARa, P, y)
Liver X receptors (LXRa, P)
Farnesoid X receptor (FXR)
Constitutive androstane receptor (CAR)
Benzoate X receptor (BXR)
Steroid xenobioticlpregnane X receptor
(SXR, PXR)
Retinoids
niques to demonstrate that RXR and RAR
bind to the 5' and 3' half-sites, respectively, of
a DR5 response element (132). The group of
ERa Chambon published similar findings and also
demonstrated that RXR occupies the 5' half-
30
site of a DR2 element (133), and this general
31
29 polarity was confirmed in crystallography
- studies in which RXR.thyroid hormone recep-
tor complexes were shown to bind with a
similar orientation to a DR4 thyroid response
element (134). Interestingly, however, Kuro-
kawa and co-workers (135) discovered that
this binding order was reversed on a DR1 ele-
ment with RAR and RXR binding to the 5' and
3' half-sites, respectively, resulting in a tran-
scriptionally inactive complex that responds
to neither trans-RA nor 9-cis-RA (see below).
7 FUNCTIONAL DOMAINS OF RARs
AND RXRs
The U s and RXRs, like other NRs, are com-
prised of a highly modular domain structure
that includes autonomous DNA and ligand
binding domains and as well as regions of the
proteins that function in both transcriptional
activation and silencing (Fig. 7.20). These
functional domains are described in detail in
the sections that follow.
7.1 RAR and RXR DNA Binding Domains
The DNA binding domains (DBDs) of mem-
bers of the NR family are, in general, highly
conserved, displaying approximately 60%
identity at the amino acid level across the en-
Orphan Receptors
Chicken ovalbumin upstream promoter-transcription
factors (COUP-TFI, 11,111)
Hepatic nuclear factor-4 (HNF-4)
NGFI-B and related proteins
7 Functional Domains of RARs and RXRs
I
Receptor function { H2N { COoH
DNA binding
Ligand binding
Dimerization
Ligand-dependent transcriptional
activation function core (AF2)
Nuclear localization signal
Ligand-independent transcriptional
activation function (AF1)
Silencing function
Figure 7.20. Nuclear receptor domain structure.
tire family. The DBD of both RARs and RXRs,
and of all NRs, is composed of approximately
66-80 amino acids that are organized into two
C,C, zinc finger motifs (see Figs. 7.16 and
7.17), each of which tetrahedrally coordinates
one zinc ion (134, 136). These two zinc fingers
fold into a single structural motif comprised of
two a helices, the recognition and support he-
lices that are located on the knuckle on the
carboxyl side of each zinc finger (134, 136).
The two a-helices play very different roles in
the function of the receptors: the recognition
helii sits in the major groove of DNA and
makes specific contacts with discriminating
nucleotides within the response element; the
support helix is oriented perpendicularly to
the recognition helix and does not contact
DNA at all (134,136). Rather, the support he-
lii makes specific protein-protein contacts
with and effectively buttresses the recognition
helix into the major groove. This concept is
illustrated schematically in Fig. 7.21. Bio-
chemical (137,138) and crystallographic (134,
136) studies have revealed that three amino
acids within the recognition helix are crucial
for response element discrimination by most
NRs.
7.2 Ligand Binding Domain
Although the ligand binding domains (LBDs)
of both W s and RXRs bind 9-cis-RA with
high affinity, the amino acid sequence identity
between the two proteins within the LBDs is
surprisingly very low (Table 7.3). Indeed, the
RAR LBD is as related in amino acid sequence
identity to that of RXR as the receptor is to
estrogen receptor a (approximately 30%; see
341
-
-
-
Table 7.3). The LBDs of RAR and RXR family
members have also been studied extensively
by biochemical means and by crystallography
(139-148). The picture that has emerged from
this large body of work suggests that the over-
all structural organization of the LBD of RARs
and RXRs is highly conserved LBD (142, 149),
even if the primary amino acid sequence is not
(see above). The structurally unique NR LBD
is comprised of 12 a-helical bundles (HI-H12;
Fig. 7.22) that sandwich a short p-turn (149).
This domain is arranged into three layers
forming what has been called an anti-parallel
"a-helical sandwich" (142). In addition to con-
ferring ligand binding, the ~ 2 2 0 to
- 240-ami-
no acid LBDs of all RAR and RXR family .
members also harbor a major dimerization in-
terface (homodimerization and heterodimer-
ization) that is comprised of amino acids from
H7, H9-Hll, and portions of the intervening
loops (140,141). The RAR and RXR LBDs also
confer all of the ligand-dependent transcrip-
tional activation potential of the correspond-
ing receptors, which has been referred to as
the AF2 activity (150). However, AF2 cannot
be described simply in linear sequence, but
rather is formed by the agonist-induced juxta-
position of several helices within the LBD (see
below).
7.3 Apo-Receptors Interact with
Transcriptional Corepressor Complexes
Essentially, the basis of retinoid receptor sig-
naling involves differential interaction of the
apo- and holo- forms of the receptors with sev-
eral, specific pools of nuclear proteins. For ex-
ample, in the absence of an agonist, apo-
Figure 7.21. Schematic representation of DNA binding domains. Structures of DNA-binding com-
plexes involving RXR and their dimer interfaces. The overall structures are shown on the left, with
close-up views of the protein-protein interactions shown on the right. Dotted blue lines indicate
hydrogen bonds between proteins or between proteins and the DNA spacing. The dotted surface
indicates complementary van der Waals interactions. DNA sequences (cyan) are shown with their 5'
ends pointing up, with base pairs belonging to the spacing element of the DRs shown schematically in
red. In each case, protein-protein contacts are formed directly over the minor groove of the spacing,
with several protein-DNA phosphate contacts stabilizing the assembly. The interacting amino acid
side-chains are shown in green, with nitrogen atoms indicated in blue and oxygen atoms indicated in
red. (a and b) The RXR-TR DBD heterodimeric complex with DR4; (c and d) the RXR DBD ho-
modimeric complex with DR1, and (e and f ) the RXR-RAR DBD heterodimeric complex with DR1.
Reprinted with permission from Curr. Opin. Struct. Biol., 1 1 , 3 3 3 8 (2001). See color insert.
 tional Domains of RARs and RXRs 343

gure 7.22. Schematic representation of apo- and holo-RXR ligand binding domain. Helices 1-12
1-H12) are indicated. Helices indicated in yellow and red represent the apo- and holo-forms of the
:eptor, respectively, whereas helices indicated in blue and green are positioned similarly in both
ms of the receptor. The arrows represent movement of the helices to accommodate 9-cis-RA in the
lding pocket (indicated). This figure was kindly supplied by Dr. Pascal Egea. See color insert.

L4R.RXR complexes are known to bind to
INA response elements loosely and recruit an
,TP-dependent, chromatin remodeling com-
lex known as ISWI to the template (151).
3WI-mediated remodeling of chromatin
eems to allow apo-RAR.RXR complexes to
ind to the DNA response element much more
ightly (151). Presumably, apo-RAR.RXR
complexes can then interact with and recruit
transcriptional corepressor proteins, such as
nuclear receptor corepressor (NCoR) and si-
lencing mediator of retinoid and thyroid re-
ceptors (SMRT) to the template (152, 153).
NCoR and SMRT, in turn, recruit histone
deacetylase complexes (HDACs) to the apo-
RAR.RXR heterodimeric complexes bound to

a response element in the promoter region of
a RA target gene (154). HDAC-mediated
deacetylation of specific lysine residues found
in multiple histones is believed to increase the
affinity of the particular histone for the DNA
template and, thus, favor the formation of
more densely packed chromatin that is gener-
ally less likely to be transcibed (155). Ten
HDACs encoded by different genes have been
isolated to date, and genetic evidence suggests
that there may be additional members of the
HDAC family that have not been described
(156-162). Thus, apo-RAR.RXR complexes
can repress the expression of at least some tar-
get genes in a HDAC-dependent manner, and
this inhibition can be relieved by inhibitors of
HDACs such as trichostatin A or valproic acid
(163). This has clinical significance because
some leukemias respond more favorably to a
combination of a retinoid and HDAC inhibitor
than to either drug alone (163, 164).
7.4 Holo-RAR.RXR Complexes Interact with
Two Classes of Transcriptional Coactivators
Agonist binding to RAR and/or RXR induces a
rather substantial conformational change in
the LBD of the receptors (Fig. 7.22). This con-
formational change is characterized by the fol-
lowing: (1) a large rearrangement involving
HI1 and H12; (2) bending of helix 3; and (3)
rotation of the omega loop separating helices1
and 213. The resultant conformation of the
holo-recevtor is not favorable for interaction
with transcriptional corepressors such as
NCoR or SMRT; thus, agonist binding-in-
duced conformational change promotes disso-
ciation of the entire corepressor complex as-
sembled on the promoter of RA target genes.
The repositioned helix 12 is generally be-
lieved to form the "lid" of the ligand-binding
pocket (143, 144, 165). The agonist binding-
induced rearrangement of helix 12 also juxta-
poses the core of AF2, which is found in helix
12, with amino acids in helices 3,4,and 5 (143,
144), creating a shallow hydrophobic groove
competent for interaction with a second class
of proteins known as transcriptional coactiva-
tors. Interaction of a RARsRXR complex with a
transcriptional coactivator requires the ago-
nist-induced juxtaposition of helices 3, 4, 5,
and 12 of the receptor with a complementary
a-helical structure in the coactivator protein
Retinoids
comprised of the consensus sequence LXXLL,
where L corresponds to a leucine residue and
X can be any non-helix breaking amino acid.
The canonical LXUL-containing a-helix of
transcriptional coactivators binds directly to
the shallow hydrophobic groove in the recep-
tor formed by the agonist induced conforma-
tional change (166-170). Thus, agonist bind-
ing-induced RAR and/or RXR conformational
change serves two purposes: (1)to induce dis-
sociation af the corepressor complex resulting
in a relief of transcriptional repression and (2)
to promote interaction of the receptor with
transcriptional coactivator proteins resulting
in transcriptional activation.
Transcriptional coactivator proteins that
interact preferentially with activated nuclear
receptors can be divided into two general fam-
ilies, each of which contain multiple subfami-
lies. Transcriptional coactivators harboring
histone acetyl transferase (HAT) activity in-
clude p300lCBP (171-174), PICAF (175-177),
and the p160 family of proteins: SRC-11
NCoA-1(167,173,178), TIF2/GRIPl/NCoA-21
SRC-2 (167,179,180),and RAC3/AIBl/PCIP/
ACTRISRC-3 (167, 181-183). By covalent
modification (acetylation) and subsequent de-
condensation of chromatin, transcriptional co-
activators possessing HAT activity are be-
lieved to enhance the access of the RNA
polymerase I1 (Pol 11)complex to the template
(184).In addition to interactingwith activated
nuclear receptors, p160 and p300lCBP pro-
teins interact with each other and the latter
interact with PICAF' (175-177). Recently, a
family of histone arginine methyltransferases
has been identified that interact specifically
with p160 family members (185,1861,suggest-
ing that the multi-protein, chromatin-modifjr-
ing machinery assembled on the promoter of
target genes is large and complex.
Interaction of the HAT coactivator complex
with the ligand-activated receptors is appar-
ently transient as dissociation is believed to
occur as a result of acetylation of one or more
coactivators on lysine residues adjacent to the
signature LXXLL motif (187). Subsequently,
the activated receptor presumably recruits a
second class of multi-protein, transcriptional
coactivator complexes to the template, and
this latter complex, referred to as the thyroid
hormone receptor-associated protein (TRAP)
8 Future Perspectives
complex, seems to be similar to the SRB- and
MED-containing cofactor (SMCC), vitamin D
receptor-interacting protein (DRIP) and the
yeast Mediator (188, 189) complexes. Because
the TRAP or TRAP-like complexes harbor
components of the Pol I1 machinery and/or re-
cruit the Pol I1 complex to the template, for-
mation of the transcriptional preinitiation
complex and subsequent transcription are fa-
cilitated (190, 191). It should be noted that
these later steps of RAReRXR-mediated tran-
scriptional activation appear to require the re-
cruitment of another protein complex to the
template, the SWIISNF complex, which seems
to be facilitated by the presence of activated
receptors, either by direct interaction or inter-
action of the SWIISNF complex with acety-
lated histones (151, 192). SWIISNF, which
also harbors an ATP-dependent chromatin re-
-
modeling activity, seems to function by dis-
placing histones in the promoter region of the
target gene, thus, facilitating tight binding of
the basal transcription factors to the tem-
plate-a prerequisite for formation of the
preinitiation complex and transcriptoinal ini-
tiation (192).
In summary, transcriptional activation me-
diated by RAR.RXR complexes is a complex,
multistep process that is not entirely under-
stood at the molecular level. However, it is
clear that agonist-induced, RAR.RXR confor-
mational change, which is crucial for dissocia-
tion of the corepressor complex and induction
of a receptor conformation that facilitates in-
teraction of the receptor
- with two distinct
classes of coactivator proteins, the HAT and
TRAP proteins, plays a central role in the sig-
naling pathways of RAR.RXR complexes lead-
ing to transcriptional activation.
7.5 "Ligand-Independent" Transcriptional
Activation, AF1, of RARs and RXRs
he amino terminal of RARs and RXRs harbor
hat is known as a ligand-independent tran-
scriptional activation function (Fig. 7.20). AF1
operates and synergizes with AF2, the li-
d-dependent transcriptional activation
nction formed by the juxtaposition of helices
thin the LBD (see above) (106, 150, 193).
owever, AF1 of retinoid receptors was de-
fined in artificial systems (by fusing this re-
gion to a heterologous DNA binding domains)
345
and may not represent real life. Clearly, AFls
contribute to the global activation potential of
retinoid receptor heterodimeric complexes,
yet the transcriptional activation activity of
these complexes is dependent on ligand, ren-
dering the "ligand-independent" activity of
AF1 entirely dependent on ligand in uiuo
(142), as has been described for other nuclear
receptors (194).
8 FUTURE PERSPECTIVES
The present and future efforts of medicinal
chemists in the development of novel retinoid
compounds are discussed in this section. This
body of work should ultimately lead to recep-
tor and receptor subtype-selective agonists
and antagonists and novel compounds that
block proliferative signaling processes.
8.1 Receptor- and Receptor
Subtype-Selective Retinoids
As discussed in a previous section, the sys-
temic and topical toxicity, as well as the ter-
atogenicity of retinoids, limits the clinical use-
fulness of these compounds. The general
opinion of both basic scientists and clinicians
is that these toxicities may be mitigated by the
development of receptor subtype-selective
retinoids. Although this may ultimately be
proven to be true, there is currently little fac-
tual basis for this opinion, at least with respect
to the RAR subtypes, because it has been quite
difficult to separate clinical efficacy from tox-
icity. Nonetheless, this remains a noble goal
- in
the continued development of receptor sub-
type-selective retinoids.
Of the seven retinoids currently approved
by the FDA for clinical use, all are agonists,
and most (adapalene, acitretin, isotretinoin,
tazarotene, and tretinoin; see Table 7.1) are
selective RAR agonists. Of these compounds,
adapalene and tazarotenic acid (the active
form of the pro-drug tazarotene) are selective
agonists for RARp and RARy, (195-1971,
whereas the other compounds activate all
RAR subtypes with roughly similar potencies.
Alitretinoin (9-cis-retinoic acid) is a pan-ago-
nist that activates all RAR and RXR sub-
types and bexarotene is a selective, RXR ago-
nist that harbors some RAR agonist activity
(198-200).

Table 7.5 Discriminatory Amino Acids in the Ligand Binding Pockets
of RAR Family Members
Receptor LBD Helix 3
RARa Ser232
RARP
MY
These amino acids, which are positioned within the ligand binding pocket of the indicated RARs, likely confer ligand
specificity.
The LBDs of RARa, P, and y are highly
related (Fig. 7.16). Moreover, there are only
three divergent amino acid residues in the li-
gand binding pockets of the RAR subtypes
(Table 7.5) (2011, rendering the development
of RAR subtype-selective ligands a challenging
endeavor. Nonetheless, substantial progress
has been made by several groups in the design
of RAR subtype-selective agonists (Fig. 7.23).
Until recently, these efforts were made largely
by empiricism, and in some cases, based on
molecular modeling algorithms. However, me-
dicinal chemists may now use the knowledge
of the three-dimensional structures of the
RAR and RXR ligand binding pockets in the
development of receptor subtype-selective
retinoids, and this will forever transform ra-
tional, retinoid drug design.
Many of the known RAR subtype-selective
ligands described below contact one or more of
the three discriminatory amino acids that line
the ligand binding pocket of the corresponding
receptor. For example, Moras and colleagues
found that Met272of RARy adopts different
ligand-induced conformations depending on
the presence of a hydrogen bond between its
sulfur atom and the corresponding ligand
(146). Similar discriminatory mechanisms
seem likely for other RARs rendering the fu-
ture development of RAR subtype-selective
agonists possible.
AM80 and AM580 (Fig. 7.23) are prototypic
compounds that activate RARa at concentra-
tions roughly 10- to 100-fold lower than those
required for activation of either RARp or
RARy (202). In general, however, it has been
more difficult to separate RARP from RARy
agonism even though these receptors differ in
two of the three discriminatory amino acids
that line their respective ligand binding pock-
ets (Table 7.5). Two somewhat RARP-selective
compounds have been reported, CD417 and
Retinoids
LBD Helix 5 LBD Helix 11
~l~~~~
~ l e ~ ~ ~ Val3"
Met272
CD2019 (203), but the degree of RARP selec-
tivity reported for these compounds is not
overwhelming. As RARy is highly expressed in
skin and is thought to mediate mgny of the
therapeutic effects of topical retinoids in the
treatment of dermatological disorders, such as
psoriasis and acne, several RARy-selective
agonists have been developed including
SR11254 (204), CD437 (203,2051, BMS184394
(203, 206, 2071, and CD666 (203). The struc-
tures of all of these receptor subtype-selective
compounds are shown in Fig. 7.23.
Antagonists of RARs have been developed,
and the prototype of these compounds is the
Ma-selective Ro41-5253 (Fig. 7.24) (208).
In addition, other novel RAR antagonists have
been developed (209,210). The clinical useful-
ness of these compounds is presently un-
known, but RAR antagonists have proven to
be very useful as tools for the dissection of the
retinoic acid signaling pathways in both cells
(201) and in animals (211). One may envision
that these receptors may work in one of two
mutually exclusive mechanisms. Firstly, the
antagonists may simply occupy the ligand
binding pocket and thereby compete with en-
dogenous agonists (trans- or 9-cis-RA, for ex-
ample). In this case, the antagonist would ef-
fectively lock the receptor in the apo-form that
is associated with transcriptional corepressor
proteins such as NCoR and SMRT (see above).
Alternatively, RAR antagonists may induce
repositioning of receptor helix 12 away from
the surface of the protein generating a novel
conformation of the receptor. In this case, he-
lix 12 would be unable to contribute to forma-
tion of the shallow hydrophobic groove that is
required for interaction with the LXXLL motif
of transcriptional coactivators (see above).
This mode of action would be consistent with
crystallographic studies that revealed that the
binding of the retinoid antagonist BMS614 to
 Compound Receptor
selectivity

RARy

Figure 7.23. RAR subtype-selective ligands.


Figure 7.24. RAR antagonist and inverse agonist.
the RARa LBD results in the repositioning of
helix 12 into the "antagonist" conformation
(140) similar to that of estrogen receptor
(ER)-P bound by the antiestrogen raloxifene
(212) or the partial agoinst genistein (213) and
ERa bound by the antiestrogen tamoxifen
(214). In these examples, binding of antago-
nists (or the partial agonist genistein) causes
juxtapositioning and subsequent interaction
of helix 12 with structural motifs in helices 3
and 4 that contribute to the coactivator inter-
action interface. In contrast to the agonist-
bound form of the receptors, however, the an-
tagonist-induced repositioning of helix 12 does
not enhance interaction of the receptors with
any of the known transcriptional coactivator
proteins possessing LXXLL motifs.
Chandraratna and colleagues at Allergan
have developed a novel class of compounds
possessing pharmacological properties that
are distinct from both agonists and antago-
nists. The prototypic compound in this class is
AGN193109 (215,216) (Fig. 7.24). In contrast
to classical agonists that induce dissociation of
RAR.NCoR complexes, AGN193109 increases
the affinity of the receptor for this corepressor
(215,216),and as such, is known as an inverse
agonist. Although AGN193109 is selective for
RARy, the property of inverse agonism, and
therefore, the development of inverse ago-
Retinoids
nists, is conceptually possible for other recep-
tor subtypes. As is the case for antagonists,
however, the clinical usefulness of such com-
pounds is presently unknown. Nonetheless,
compounds such as inverse agonists and an-
tagonists are great tools that allow retinoid
biologists to dissect the retinoid signaling
pathways in various contexts.
Whereas it has been relatively straightfor-
ward to develop compounds that discriminate
between RARs and RXRs using, for example,
trans-RA as the prototypic pharmacophore
that is specific for RAR subtypes, the develop-
ment of RXR-selective compounds has also
progressed as a result of the synthetic efforts
of a number of medicinal chemists. RXR-selec-
tive retinoids (Fig. 7.25) include bexarotene
(198, 199), SR11237 (2171, SR11246 (200,
204), LGD100268 (199), and SR11345 (M.
Dawson, X.-K. Zhang, and M. Leid, unpub-
lished data, 2000). The degree of RXR sub-
type-selectivity of these compounds is un-
known.
One major obstacle for the development of
RXR subtype-selective compounds is that the
amino acid residues lining the ligand binding
pockets of RXRa, P, and yare identical (143).
However, these RXR subtypes do harbor dif-
ferences in so-called "second layer residues,"
those amino acids that do not contact the li-
gand directly, but nonetheless must undergo
side chain conformational alterations to ac-
commodate ligand in the binding pocket (143,
146). Thus, it is conceivable that substitutions
in second layer residues of RXR subtypes may
be exploited in the development of RXR sub-
type-selective ligands.
8.2 "Gene-Specific" Retinoids
Heterodimeric RAFi.RXR complexes bind to
and regulate target gene transcription from
DR1-, DR2-, and DR5-type response elements
(see previous sections). Thus, it is conceivable
that retinoids could be developed that would
selectively activate a RAReRXR complex
bound to a DR5 RARE, for example, but not
the same complex bound to either DR1 or DR2
RAFiEs, or other, unknown response ele-
ments. It has been suggested that such a phe-
nomenon may be caused by the existence of
alternative conformations of the RAR.RXR
heterodimeric complexes bound to these dif-
8 Future Perspectives
Bexarotene
Figure 7.25. RXR-selective agonists.
ferently spaced response elements, differing
promoter contexts, or both (218). Although
this is an appealing idea in principle and some
evidence derived from artificial systems has
been provided to support this concept, con-
, vincing data are lacking. Nonetheless, as more
i information becomes available regarding the
: structure of heterodimeric complexes bound
, to differently spaced response elements and
, the mechanisms underlying RAR.RXR-medi-
ated transcriptional activation, it may be con-
ceivable that so-call "gene-specific" retinoids
could be developed.
8.3 Anti-AP1 Activity of Retinoids
Ligand-activated RAR-RXRcomplexes antag-
onize transcriptional activation mediated by
the proto-oncogene transcription factor com-
plex known as AP1, and the converse is also
true. AP1 antagonizes RAR.RXR-mediated
transcriptional activation (173, 219). Activa-
tion of RARs by classical retinoid receptor
agonists, such as trans-RA, results in two
events, activation of transcription mediated
by RAR.RXR complexes and antagonism of
AP1-mediated transcriptional activation that
may be mediated by RAReFtXR or RAR alone
(220-223). As AP1 is classically associated
with cellular proliferation and inflammatory
responses, retinoid receptor-mediated antago-
nism of AP1-mediated transcriptional activa-
tion could be exploited therapeutically in the
treatment of proliferative diseases and inflam-
matory diseases such as arthritis. The Dawson
group was the first to develop so-called anti-
AP1 retinoids, the prototypes of which are
SR11238(Fig. 7.26) and SRll302 (219),which
do not modulate the transcriptional activation
function of any of the known RARs or FtXRs.
The molecular basis of action of compounds
such as SR11238 is unknown, but two mecha-
nisms have been proposed to account for this
form of transcriptional interference: (1)com-
petition between retinoid receptors and AP1
for a common transcriptional coactivator,
such as CBP (173), and (2)retinoid receptor-
mediated inhibition of AP1 assembly (223).
Clearly, development of useful and selective
retinoids that induce the receptors to inhibit
AP1-mediated transcriptional activation with-
out affecting the transcriptional activation
Figure 7.26. Anti-APl retinoid, SR11238.
350
function of the receptors remains an impor-
tant goal in the retinoid field.
8.4 0 4 3 7 : An Atypical Retinoid
CD437, 6-[3-(1-adamanty1)-4-hydroxyphenyll-
2-naphthalenecarboxylicacid (AHPN; see Fig.
7.23), was originally described as a selective,
M y agonist that may be useful in the treat-
ment of retinoid-sensitive dermatological con-
ditions (203, 205). However, more recently,
Fontana and colleagues have discovered that
this retinoid induces cell cycle arrest and sub- tivate transcription. The most important of
sequent apoptosis in a number of cancer cell
lines (224-232) through a mechanism that
does not seem to involve any of the known
RAR or RXR subtypes (229). CD437/AHPN
even induces growth arrest and apoptosis in
cancer cell lines that are resistant to growth
inhibition induced by classical retinoids such
as trans-RA (229). Neither the putative recep-
tor for CD437 nor the pro-apoptotic mecha-
nism have been identified.
Again, however, the toxicity of CD4371
AHPN is a problem in a clinical sense, and this
toxicity seems likely to result from activation
of RARs. Thus, substantial efforts have been
devoted to the development of CD437lAHPN-
like compounds that maintain pro-apoptotic
activity with reduced ability to activate classi-
cal retinoid receptors. The first such com-
pound is MM11453, the 3-chloro analogue of
AHPN (232). MM11453 was found to inhibit
the growth of both retinoid-resistant and ret-
inoid-sensitive cancer cell lines with equal po- of highly insulin-resistant patients harbor a
tency (232). It is highly likely the additional
compounds like MM11453 will be developed
for the treatment of proliferative disorders.
8.5 Novel Uses for RXR-Active Compounds
As described above, RXR heterodimerizes
with several other members of the nuclear re-
ceptors family. This implies that RXR,
through these diverse, heterodimeric interac-
tions, may regulate the expression of a very
large number of genes. However, RXR does
not seem to be competent to active transcrip-
tion in the context of VDR.RXR, TR.RXR, or
M . R X R complexes bound to DR3-, DR.-, or
DR5-type response elements, respectively
(135,233). In the case of RAR-RXRcomplexes
(234, 235) and probably also TRsRXR com-
Retinoids
plexes (236),RXR can activate transcription if
the other subunit of the heterodimer is bound
by either agonists (235, 236) or an antagonist
(234). This finding suggests that the RXR het-
erodimeric partner allosterically regulates the
function of the RXR LBD and is known as the
"RXR subordination" model (210) in that RXR
is dependent on (subordinate to) activation of
the its heterodimer partner for its own activa-
tion.
In other heterodimeric contexts, known as
"permissive heterodirners," RXR is able to ac-
these in a clinical sense are probably
RXR.PPAR (237-239) and RXR.LXR (240)
heterodimers. The ability of RXR agonists to
activate RXR.PPARy heterodimers is ex-
tremely important in the treatment of type I1
diabetes (non-insulin dependent diabetes), a
disease of epidemic proportions in the United
States and worldwide. For example, thiazol-
idinedione antidiabetic drugs are becoming in-
creasingly used in the treatment of type I1 di-
abetes. These drugs are PPARy agonists that
activate RXR.PPARy heterodimeric com-
plexes that, in turn, regulate the expression of
genes encoding proteins that improve glucose
tolerance by enhancing the cellular sensitivity
to insulin. Administration of RXR agonists to
diabetic and obese mice also improves glucose
tolerance, and the co-administration of RXR
agonists with a thiazolidinedione results in
synergistic improvements in several diabetic
parameters (241, 242). Interestingly, subsets
mutation(s) in the PPARy locus that renders
these patients resistant to thiazolidinedione
therapy, yet these patients remain sensitive to
the glucose-lowering effects of RXR agonists
(237). Thus, the continued development of
RXR-selective ligands for the treatment of
type I1 diabetes, as monotherapy or in con-
junction with thiazolidinediones, will cer-
tainly be pursued in the future.
Another interesting application for RXR-
selective agents may be in the treatment of
hypercholesterolemia. Mangelsdorf and col-
leagues have recently discovered that the RXR
agonist LG100268, acting through RXR.LXR
heterodimeric complexes, transcriptionally
induces the expression of the gene encoding
the ABCl protein, which mediates reverse
cholesterol transport (240). In intestinal cells,
this protein transports cholesterol into the lu-
men of the intestine, effectively reducing cho-
lesterol uptake by the cell and ultimately low-
ering blood cholesterol levels. In the case of
RXR agonists, this effect is amplified because
RXR agonists (rexinoids) also decrease bile
acid secretion through the activation of
RXRFXR heterodimeric complexes (243,244)
(Table 7.4). Because bile acids are important
vehicles for cholesterol dissolution and gener-
ally promote cholesterol absorption in the gut,
the effect of RXR-mediated induction of ABCl
expression has an even more dramatic effect
on serum cholesterol levels. In addition, the
ABCl protein mediates cholesterol efflux from
macrophages that contribute to atheroscle-
rotic processes (240). The development of ad-
ditional RXR-selective ligands will clearly be
at the forefront of retinoid biology in the fu-
ture. However, there are problems with rexi-
noid therapy, including disruptions in triglyc-
eride levels and possibly obesity, the latter of
which is controlled, at least in part by
RXRPPARy complexes (245, 246). Addition-
ally, systemic administration of & RXR ago-
nist has been associated with reversible, cen-
tral hypothyroidism, most likely by activation
a TR.RXR heterodimeric complex that re-
presses thyrotropin secretion (236).
8.6 Summary and Perspective
Retinoids, vitamin A derivatives, play essen-
tial roles throughout organismal life. Al-
hough the mechanisms underlying the pleio-
ropic effects of retinoids are not completely
derstood at the mechanistic or cellular
evel, naturally occurring retinoids and syn-
thetic derivatives are being increasing used in
nical medicine. The past 5 years have seen a
amatic increase in the use of retinoids to
eat a variety of diseases. In addition, several
w uses of retinoids are presently being in-
stigated. The next 5 years will likely see in-
eased development of rexinoids, RXR- and
subtype-selective compounds to treat a
ety of diseases that were not previously
ought to be responsive to retinoid therapy.
ow more than ever, retinoid chemistry is
ised to make substantial contributions to
human health through the design and synthe-
sis of novel compounds that act at both RARs
and RXRs.
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Contents
1 Introduction, 361
1.1 What Is a Vitamin?, 361
1.1.1Vitamins Are Naturally Occurring, 361
1.1.2 Vitamins Are Essential Because They
Are Not Produced by Human
Biochemical Pathways, 361
1.1.3 Organic, 361
1.1.4 Normal Constituent of the Diet, 362
1.1.5 Vitamins Are Required in Minute
Amounts, 362
1.1.6 Vitamins Are Required to Maintain
Normal Biochemical Functions of the
Tissues, 362
1.2 Causes of Vitamin Deficiencies, 362
2 Dietary Reference Intakes, 362
2.1 Determination of a Vitamin Dose, 362
2.2 Methods to Determine a Valid Dose
of a Vitamin, 362
2.2.1 Extrapolate from Animal Studies, 362
2.2.2 Metabolic Balance Studies in Humans,
363
2.2.3 Compare Nutrient Intake in Areas
with and without the Deficiency
Disease, 363
2.2.4 Saturation of Biochemical Function, 363
2.2.5 Serum Levels, 363
2.3 Dietary Reference Intakes (DRIs) (3,4), 363
2.3.1 Introduction, 363
2.3.2 How Do the DRIs Differ h m RDAs?, 363
2.3.3 Uses of Dietary Reference Intakes, 363
2.3.3.1 Estimated Average
Requirement (EAR), 363
2.3.3.2 Recommended Dietary
Allowance (RDA), 363
2.3.3.3 Adequate Intake (AI), 368
2.3.3.4 Tolerable Upper Intake Level
(UL), 368
3 Vitamins, 368
3.1 Retinol Witamin A) Family (91, 368
3.1.1 Chemistry, 368

3.1.2 Uptake and Metabolism (Fig. 8.3), 369
3.1.3 Biochemical Functions and Deficiency,
370
3.1.4 Dosage Forms, 370
3.1.5 Hypervitamininosis A, 370
3.1.6 Hypercarotenosis, 371
3.1.7 Dietary Reference Intakes (as Retinoic
Acid Equivalents), 371
3.1.8 Pharmacologically Active Retinoids, 372
3.1.8.1 Retinoid and Retinoid-like
Drugs Used in the Treatment of
Acne (Fig. 8.7) (12), 372
3.1.8.2 Drug Based on the Retinoid
Structure Used to Treat
Psoriasis (Fig. 8.81, 373
3.1.8.3 Retinoids Used in the
Treatment of Malignancies (Fig.
8.9), 374
3.2 Vitamin D Family (15), 374
3.2.1 Chemistry, 374
3.2.2 Vitamin Uptake and Metabolism, 375
3.2.3 Biochemical Function, 376
3.2.3.1 Calcium Regulation, 376
3.2.3.2 Vitamin D Analogs Used in
Chronic Renal Failure, 377
3.2.3.3 Cell Division, 377
3.2.4 Dosage Forms, 379
3.2.5 Hypervitaminosis D, 379
3.2.6 Dietary Reference Intakes, 379
3.2.7 Drug Interactions, 379
3.3 Vitamin E Family (Tocopherols
and Tocotrienols) (33), 380
3.3.1 Chemistry, 381
3.3.2 Uptake and Metabolism, 381
3.3.3 Biochemical Function, 383
3.3.4 Deficiencies, 383
3.3.5 Hypervitaminosis E, 384
3.3.6 Dosage Forms, 384
3.3.7 Dietary Reference Intakes (based on
d-a-Tocopherol), 384
3.4 Vitamin K Family (381,385
3.4.1 Chemistry, 385
3.4.2 Vitamin K Uptake and Metabolism, 386
3.4.3 Vitamin K Biochemistry and Defi-
ciency, 386
3.4.4 Causes of Vitamin K Deficiency, 387
3.4.5 Hypervitaminosis K, 387
3.4.6 Dietary Reference Intakes, 387
3.5 Thiamine (Vitamin B,) (43), 387
3.5.1 Chemistry, 388
3.5.2 Uptake and Metabolism, 388
3.5.3 Thiamine Deficiencies, 389
3.5.4 Hypervitaminosis Thiamine, 389
3.5.5 Dietary Reference Intakes, 389
3.6 Riboflavin (Vitamin B,) (46), 392
Vitamins
3.6.1 Chemistry (Fig. 8.281, 392
3.6.2 Riboflavin Uptake and Chemistry (Fig.
8.281, 392
3.6.3 Metabolic Role, 392
3.6.4 Riboflavin Deficiency, 392
3.6.5 Hypervitaminosis Riboflavin, 392
3.6.6 Dietary Reference Intakes, 392
3.7 Niacin (Nicotinic Acid)/Niacinamide
(Nicotinamide) (47), 392
3.7.1 NiacinNiacinamide Chemistry, Uptake
and Metabolism, 394
3.7.2 Niacin Deficiency, 394
3.7.3 Hypervitaminosis Niacin, 395
3.7.4 Dietary Reference Intakes, 397
3.8 Vitamin B, Family, 397
3.8.1 Uptake and Metabolism, 397
3.8.2 Pyridoxal Phosphate Biochemistry
(58), 397
3.8.3 Vitamin B, Deficiency, 398
3.8.4 Vitamin-Drug Interactions (60), 399
3.8.5 Hypervitaminosis Pyridoxine, 400
3.8.6 Dietary Reference Intakes, 400
3.9 Pantothenic Acid (651,400
3.9.1 Chemistry, Uptake, and Metabolic
Role, 401
3.9.2 Hypervitaminosis Pantothenic Acid, 401
3.9.3 Dietary Reference Intakes, 401
3.10 Biotin (661,402
3.10.1 Uptake, 402
3.10.2 Chemistry, 402
3.10.3 Metabolic Role, 402
3.10.4 Biotin Deficiency, 405
3.10.5 Hypervitaminosis Biotin, 405
3.10.6 Dietary Reference Intakes, 405
.
3.11 Folic Acid (67), 405
3.11.1 Chemistry, 405
3.11.2 Uptake, 405
3.11.3 Metabolic Roles, 406
3.11.4 Folic Acid Deficiency, 407
3.11.5 Hypervitaminosis Folic Acid, 411
3.11.6 Dietary Reference Intakes, 411
3.12 Vitamin B,, (Cobalamin) (721,411
3.12.1 Chemistry, 413
3.12.2 Uptake, 413
3.12.3 Biochemical Role, 413
3.12.4 Cobalmin Deficiency, 415
3.12.5 Hypervitaminosis B,,, 415
3.12.6 Dietary Reference Intakes, 415
3.13 Ascorbic Acid (Vitamin C) (771,415
3.13.1 Chemistry, 415
3.13.2 Uptake, 417
3.13.3 Metabolic Roles, 417
3.13.4 Ascorbic Acid Deficiency, 417
3.13.5 Hypervitaminosis C, 417
3.13.6 Dietary Reference Intakes, 418
1 INTRODUCTION
Vitamins are enjoying renewed popularity
with the lay public that, in some ways, mimics
the attention seen in the early part of the
twentieth century when they were first being
discovered. Paralleling the interest by the gen-
increased focus on the biochem-
tamins at the molecular level.
Some vitamins can be considered prototypes
for pharmacological agents used to treat dis-
eases that do not appear directly related to the
patient's vitamin status. Although all animals
require vitamins, the discussion in this chap-
ter focuses on humans and human biochemi-
Vitamins are complex biochemically and
functionally and may be classified many ways.
This chapter acknowledges the traditional sol-
ubility model but focuses on functionality. Un-
fortunately,the loose regulation of nutritional
products in the United States has led to mis-
leading promotion of these items to the lay
at individuals are confused as to
at can be called a vitamin. General criteria
a substance truly is a vitamin
presented. Another problem is to decide
nt actually has a vitamin defi-
in the next section, this can be
complex question. Finally, deciding how
cular vitamin should be con-
ed has moved from the familiar Recom-
ended Dietary Allowance (RDA) to four dif-
rent ways to evaluate vitamin requirements
d consumption. These are called the Dietary
ference Intakes (DRIs).
.1 What i s a Vitamin?
In general a vitamin must be a naturally oc-
olecule that is a normal con-
iet. It should be essential and
inute amounts. Finally, it is
ain normal cellular biochem-
and tissue integrity. Let us examine
in more detail.
1.1.1 Vitamins Are Naturally Occurring.
his criterion frequently is misinterpreted.
though the vitamin is a natural product,
ere is no difference in efficacy between in-
sting the all-natural or a synthetic product
as long as the natural and synthetic products
are identical. This includes stereochemistry.
L-Ascorbic acid is twice as active as the racemic
D,L-ascorbicacid. A similar statement can be
made for D-pantothenic acid and S-biotin rela-
tive to their racemic mixtures. The situation
for a-tocopherol (vitamin E) with three asym-
metric centers is more complex and is dis-
cussed in more detail later in this chapter.
1.1.2 Vitamins Are Essential Because They
Are Not Produced by Human Biochemical Path-
ways. With two exceptions, this requirement
is obvious. If a compound is required for a bio-
chemical process and our pathways cannot
produce it, it must be obtained from exoge-
nous sources. The evolutionary history of the
human species is such that we sought out
sources in our environment that contained
these essential substances, and those sub-
stances containing vitamins became our food.
One of the substances commonly treated as
a vitamin is niacin, which is synthesized from
the essential amino acid tryptophan. The ratio
is approximately 60 mg of tryptophan being
required to produce 1 mg of niacin (1). This
has led to niacin requirements being ex-
pressed as niacin equivalents (NE), based on
the amount of tryptophan in the diet. It must
be kept in mind that tryptophan is essential
and is the precursor to the neurotransmitter
serotonin in addition to being part of protein
structure. Therefore, niacin can be thought of
as tryptophan sparing.
The other exception is vitamin D,, or chole-
calciferol. Assuming adequate sunlight, chole-
calciferol is the photochemical product from
ultraviolet irradiation of 7-dehydrocholesterol
found in our skin. A significant proportion of
the world's population produces all of the
cholecalciferol it needs from this photochemi-
cal reaction. It is true that a ~hotochemical
*
reaction is not a biochemical pathway, but ex-
posure to adequate sunlight does fulfill the re-
quirement for cholecalciferol. As noted in the
discussion for this particular substance, the
compounds commonly referred to as vitamins
D, and D, are not normal constituents of most
human diets.
1.1.3 Organic. Trace elements are not
properly called vitamins. Therefore, iron, zinc,

magnesium, manganese, chromium, sele-
nium, and the other trace elements that are
obviously obtained from food sources are es-
sential, but they are not vitamins.
1.1.4 Normal Constituent of the Diet. With
the exception of cholecalciferol,all of the other
compounds that we treat as vitamins are nor-
mal constituents of our diets, with most found
in more than one food group. The situation
with cholecalciferol is such that, except for the
small part of the world's population that ob-
tains its protein from marine species, neither
cholecalciferol nor ergocalciferol from irradia-
tion of the plant sterol ergosterol, is a normal
component of foods.
1.1.5 Vitamins Are Required in Minute
Amounts. This is arbitrary, but ranges from
2.0 pg g) for cyanocobalamin to 90 mg
for ascorbic acid. Other essential nutrients in-
cluding the essential amino acids and fatty ac-
ids generally are required in larger amounts.
For example, the National Research Council's
recommended daily intake of amino acids
ranges from a low of 245 mg for tryptophan to
980 mg for phenylalanine and tyrosine (com-
bined) and leucine (2). Note that the value for
tryptophan provides only about 4 mg of niacin,
assuming all 245 mg of tryptophan is con-
verted to niacin.
1.1.6 Vitamins Are Required to Maintain
Normal Biochemical Functions of the Tissues.
Most vitamins function either as a hormone1
chemical messenger (cholecalciferol), struc-
tural component in some metabolic process
(pantothenic acid), or a coenzyme (phytonadi-
one, thiamine, riboflavin, niacin, pyridoxine,
biotin, folic acid, cyanocobalamin). At least
one vitamin has more than one biochemical
role. Vitamin A as an aldehyde (retinal) is a
structural component of the visual pigment
rhodopsin and, in its acid form (retinoic acid),
is a regulator of cell differentiation. The pre-
cise biochemical functions of ascorbic acid and
a-tocopherol still are not well defined.
1.2 Causes of Vitamin Deficiencies
There are a variety of reasons why a person
might be experiencing a vitamin deficiency
Vitamins
including economics, genetics, diseased intes-
tinal tract, alcoholism, a variety of medical
conditions, lifestyle, and vitamin-drug inter-
actions. Table 8.1 contains a classification
scheme for possible causes of vitamin deficien-
cies.
2 DIETARY REFERENCE INTAKES
2.1 Determination of a Vitamin Dose
This is not easy. Ideally, a dose-response curve
could be constructed that would provide de-
fined endpoints such as an ED,, or ED,, and
doses that would cause serious toxicities, the
LD,,. The problem is defining a biological or
pharmacological endpoint. Not all vitamin de-
ficiencies have defined syndromes. Little is
known of the pharmacokinetics of individual
vitamins. In contrast with most drugs, the
body stores vitamins, sometimes several
months' supply. This should not be surprising
when one examines human history. Until
fairly recent times, humans regularly experi-
enced food plenty and food deprivation (famine)
as crops failed and animals moved away from
settlements. Causes ranged from weather to
war. To survive, the evolutionary development
of humans includes ways to store and reuse vi-
tamins. Assuming a good diet, humans have a
6- to 9-month supply of vitamin A. Humans ef-
ficiently recycle cobalamin (vitamin B,,) by
enterohepatic circulation. The intestinal flora
produce a precursor to vitamin K.
2.2 Methods to Determine a Valid Dose
of a Vitamin
Each of the methods outlined in the following
sections has significant problems in determin-
ing a dose-response curve for a vitamin. Table
8.2 lists methods that are being used to deter-
mine vitamin requirements or are under de-
velopment as possible procedures.
2.2.1 Extrapolate from Animal Studies.
This method is dependent on the species. The
vitamin requirements differ among the com-
mon laboratory animals. The classic example
is ascorbic acid, which is not a vitamin in most
animals. Humans do not exhibit specific defi-
2 Dietary Reference Intakes
ciency symptoms for a-tocopherol, biotin, and
pantothenic acid, whereas many animals do.
2.2.2 Metabolic Balance Studies in Humans.
This usually involves a specified number of
days on a defined diet in which the urine and
feces are monitored. The problem is that cor-
rections have to be made for the vitamins that
are stored. The pharmacokinetics of most vi-
tamins have not been carefully determined.
2.2.3 Compare Nutrient Intake in Areas
with and without the Deficiency Disease.
First, not all vitamin deficiencies lead to a de-
fined deficiency state. Second, rarely does one
h d an area deficient in only one nutrient.
Most common deficiencies are attributed to in-
adequate diet, which means several nutrient
deficiencies will result.
2.2.4 Saturation of Biochemical Func-
tion. A reliable biochemical indicator is re-
quired. For niacin, which NADt- or NADP+-
ntaining enzyme should be selected? Which
ansaminase will be the indicator for pyridox-
e? Which function of vitamin A should be
lected for retinol, vision in the rods or cell
rentiation? As noted from Table 8.2,
of the assays for vitamin status have
gnificant limitations to estimate reliable
2.2.5 Serum Levels. This probably is the
ost reliable, but it does require very sensi-
ve assay methods for those vitamins re-
ired in microgram (lop6)amounts. It also
uires knowledge as to how the vitamin is
ansported: free or bound to a plasma protein
on a specific transport protein. Examples of
e latter are transport proteins for vitamin A
d vitamin D,. The tocopherols will be found
in the lipoproteins (VLDL, LDL, etc.).
3 Dietary Reference Intakes (DRls) (3, 4)
e adult DRI values for each vitamin are
und in the section for that vitamin.
2.3.1 Introduction. The last official set of
rence values were the 1989 Recommended
tary Allowances (RDAs) for the United
tates and 1990 Recommended Nutrient In-
takes for Canada. These are being replaced by
the Dietary Reference Intakes, a joint effort of
the United States and Canada. The DRIs are
published by the Food and Nutrition Board of
the National Academy of Sciences National
Research Council. The expert panels are made
up of U.S. and Canadian scientists.
2.3.2 How Do the DRls Differ from RDAs?.
There is one set of reference values for both
Canada and the United States and clear docu-
mentation on how reference values are se-
lected. A goal is the promotion of nutrient
function and biologic-physical well-being. Ev-
idence concerning the prevention of disease
and developmental disorders is examined in
addition to the traditional "how much nutri-
ent is needed to prevent a deficiency symp-
tom." Data supporting food components that,
up to this time, have not been considered es-
sential nutrients will be examined. Finally,
there are recommendations for future re-
search.
2.3.3 Uses of Dietary Reference Intakes.
The DRIs consist of four components. Each
type of reference value is calculated from daily
intakes averaged over time (usually one or
more weeks). The surveys include, but are not
limited to, (1)random selection of healthy in-
dividuals and asking them to either report
what they have eaten or to maintain food dia-
ries, (2) monitoring overall food production
and consumption, and (3) correlating a de-
fined population's health status with the
group's food intake. Sometimes the results
from the surveys are correlated with the type
of assays listed in Table 8.2. The four Dietary
Reference Intakes are:
2.3.3.1 Estimated Average Requirement
(EAR). The EAR is the intake that meets the
estimated nutrient need of 50% of the individ-
uals in that group (i.e., infants, children, adult
males, adult females, pregnant women, nurs-
ing women, the elderly, etc.). It is used to eval-
uate the adequacy of nutrient intakes of pop-
ulation groups and for planning intakes for
group. It can be used in diet planning. The
EAR is based on a median rather than a mean.
2.3.3.2 Recommended Dietary Allowance
(RDA). The RDA is the intake that meets the
nutrient need of almost all (97-98%)individu-
Table 8.1 Causes of Nutrient Deficiencies
- - -

Cause Mechanism/Reason Remarks

Inadequate ingestion
usually from a poor diet
Inadequate absorption
Economic deprivation
Self-imposed reducing diets
Disease
Diseased intestinal tract
Mineral oil laxatives, which may dissolve the oil-soluble
vitamins
Ion exchange resins (colestipol, colestyramine), which
complex with the bile salts and can interfere with the
absorption of the oil-soluble vitamins
Aluminum antacids can complex with some of the
vitamins and, when used chronically, most definitely
can cause hypophosphatemia
Cystic fibrosis, which can cause fat malabsorption
(steatorrhea) attributed to inadequate production of
pancreatic lipases
Inability to purchase adequate amounts and variety of
food.
A 1200 calorie diet professionally selected from the four
major food groups (dairy, fruits and vegetables,
grains, and meat) containing no fried food nor added
sugar has been considered the least amount of food
not requiring a vitamin supplement.
This usually is attributed to loss of appetite from such
conditions as cancer chemotherapy, depression, and
eating disorders.
Examples include chronic inflammatoryconditions such
as Crohn's disease and parasites.
This could include retinol, cholecalciferol/ergocalciferol,
a-tocopherol, vitamin K from food.
A vitamin supplement can be taken 1 h before or 2 h
after taking the resin.
Aluminium antacids are no longer commonly used.
This could include retinol, cholecalciferol/ergocalciferol,
a-tocopherol, vitamin K from food, all of which are
more readily absorbed when they can be part of a
normal mixed micelle process that occurs with lipid
digestion and absorption
 (MSUD), which will respond to thiamine (vitamin B,)
supplements, and homocystinuria, which will respond
to pyridoxine (vitamin B,). The mutation lies with
apoenzyme such that the equilibrium between the
apoenzyme and the coenzyme lies to the left. To push
the reaction to the right requires additional
coenzyme.
Drug-vitamin interactions These can interfere with vitamin processing in the
intestinal tract, tie up the vitamin preventing it from
being used, or possibly promote elimination of the
vitamin. Examples include: isoniazid-pyridoxine,
phenobarbital-cholecalciferol, methotrexate-folic
acid, phenytoin-folic acid.
Increased requirements Reference dietary indices are based on an average Individuals performing more strenuous activities
above the recommended population performing average duties. Increased requiring additional intake of calories also will
daily allowances (RDA) physical activity or medical needs make these people require more nutrients including vitamins.
statistical outliers beyond the requirements of the Vitamin and caloric requirements increase for patients
"average" healthy population group. experiencing debilitating illness, including severe
burns, major surgeries, and malignancies. Nutritional
assessments are becoming a more common part of
medical treatment.
Chronic intake of alcohol Ethyl alcohol can interfere with the uptake, processing, The two most common vitamin deficiencies seen in
(alcoholism) or storage of vitamins. patients chronically consuming alcohol are folic acid
and thiamine.
Table 8.2 Methods for Estimating Vitamin Requirements
Vitamin Determination Methodology
Retinol (vitamin A) Dark adaption test
Pupillary response test
Plasma retinol concentration
Relative dose response
Calciferol (vitamins D, Serum 25(OH)D, or D,
and D,)
a-Tocopherol (vitamin E) Lipid peroxidation markers
Plasma a-Tocopherol
concentration
Vitamin K Prothrombin time
Factor VII
Plasma or serum phylloquinone
concentration
Urinary y-carboxyglutamyl
residues
Thiamine (vitamin B,) Erythrocyte transketolase
activity
Urinary thiamine excretion
Erythrocyte thiamine
concentration
Riboflavin (vitamin B,) Erythrocyte glutathione
reductase activity
Erythrocyte flavin
Urinary riboflavin excretion
Urinary excretion
Plasma concentration
Erythrocyte pyridine nucleotides
Remarks
Considered reasonably sensitive.
Data do not exist relating pupillary threshold sensitivity to retinol intake.
Insensitive to liver stores.
An initial plasma concentration is determined followed by a second
concentration a defined time after administration of a small dose of retinol.
The ratio is proportional to the liver stores of retinol.
This is considered a more accurate indicator of vitamins D status as compared
to serum vitamins D, or 1,25(OH),D.
They are not specific to vitamin E.
It does not seem to correlate with daily intake, but there is a linear relationship
seen in tocopherol-depleted subjects.
It is used to assess patients on cournadin anticoagulant therapy, but does not
appear to be a reliable measure of vitamin K status in otherwise healthy
subjects.
Even though factor VII only has a 6-h half-life, it does not appear to be
reliable.
This does respond to changes in dietary intake within 24 h.
This is considered promising.
This is considered the most accurate.
This method also is considered good.
It is not as widely used.
It is the most common assay.
Some question the sensitivity of this test because the difference between
adequacy and inadequacy is small.
Care must be exercised with the size of the dose and method of administration.
N1-Methylnicotinamide and N1-methyl-2-pyridone-5-carboxamide are the two
metabolites that are considered sensitive measures of niacin status.
N1-Methyl-2-pyridone-5-carboxamide plasma levels provide an indication of low
niacin intake by dropping below detection limits.
Erythrocyte NAD levels may replace measuring metabolites found in the urine.

~r~thro& e total blood
and
pyridoxal phosphate
Urinary pyridoxic acid
Various erythrocyte
transaminases
Tryptophan catabolites
Plasma homocysteine
Pantothenic acid Urinary excretion
Blood levels
Biotin Urinary excretion
Folic acid Erythrocyte folate
Plasma homocysteine
Serum folate
Urinary folate
Hematological status
Vitamin B,, (cobalamin) Hematological response
Serum or plasma vitamin B,,
Methylmalonic acid
Homocysteine
Holotranscobalamin I1
Ascorbic acid (vitamin C) Antioxidant functions
Cellular DNA damage
Urinary markers
. .
It changes slowly in response to changes in vitamin intake.
There &ay be r&al differences because of lower kinase activity.
It tends to reflect recent vitamin intake rather than general vitamin status.
Aspartate and alanine transaminases have been studied most extensively.
There is a lack of consensus regarding their usefulness as indicators of
vitamin B, status.
Although useful, some of the reactions affected changes in steroid hormone
status.
See discussion in the vitamin B, section.
It is strongly dependent on intake.
There is poor correlation with urinary excretion values.
There is a correlation with induced deficiencies caused by eating raw egg white.
Only the developing erythrocyte takes up folate. Therefore, this test is an
indicator of long-term status and not a measure of immediate changes in
folate status.
There is no question that elevated homocysteine decreases with folate
administration. There is not enough information to support its use for
determining DRIs.
A measure of dietary folate intake, although it is not considered a reliable
measure of folate status.
This test may underestimate folate needs.
Characteristic megaloblastic anemia develops late in folic acid deficiency.
These are the typical hemoglobin, hematocrit, and erythrocyte counts. Partial
.
resDonses are of limited values.
A problem is that serum values may be maintained at the expense of tissue
stores.
Studies need to be done to see whether it will correlate with vitamin B,, status.
This can be elevated in folate and vitamin B, deficiencies.
This protein is responsible for receptor-mediated uptake of B,, into cells.
Further work needs to be done before it is adapted for routine clinical use.
A variety of markers have been evaluated including LDL, VLDL,
malondialdehyde, hydroxynonenal, and reduced glutathione.
These have not proved useful for estimating ascorbate requirements.
Oxidized DNA is nonspecific.

als in that group. The RDA are the values
found on most vitamin products. They vary
depending on whether the product is intended
for infants, children, or adults. The RDAs can
function as a guide to achieve adequate nutri-
ent intake. By themselves, they are not gener-
ally recommended for diet planning for spe-
cific groups of individuals. Diet planning must
take into account extensive physical activity,
type of body build including lean vs. adipose
tissue, general lifestyle, and so forth. If the
sampling and endpoints are well defined, the
RDA can be calculated from the EAR.
where SDEm is the standard deviation above
the EAR.
2.3.3.3 Adequate Intake (Al). The A1 is the
average observed or experimentally derived
intake by a defined population or subgroup
that appears to sustain a defined nutritional
state, such as normal circulating nutrient val-
ues, growth, or other functional indicators of
health. It is derived from mean intakes of
groups (rather than individuals). The AI be-
comes most useful when a reliable EAR is not
available. Many times the AI probably exceeds
the EAR and possibly the RDA.
2.3.3.4 Tolerable Upper Intake Level (UL).
The UL is the maximum intake by an individ-
ual that is unlikely to pose risks of adverse
health effects in almost all (97-98%)individu-
als. It includes intake of a nutrient from all
sources (food, fortified food, water, and sup-
dements).
- Water can include fluoride and
minerals depending on the source of water.
"Tolerable" is used to "avoid implying any
possible beneficial effect." It is the amount of
vitamin that can be "tolerated" without the
person's exhibiting or experiencing adverse
reactions. The UL should not be considered
the upper dose for those who self-dose with
megadoses of vitamins.
3 VITAMINS
The order of vitamins in this chapter follows
the classical classification-based solubility: fat
soluble and water soluble. This classification
originated before there was any understand-
Vitamins
ing of the structural chemistry of the vita-
mins. There was fat-soluble A and water-solu-
ble B. Thiamine was the first vitamin (B,)
whose structure was elucidated. It is an m i n e
leading to the term vital amine and finally vi-
tamin (without the e ) . The letter designations,
for the most part, are being relegated to his-
tory. Many of the vitamins are groups of struc-
tures and, therefore, using a single letter can
be misleading. Also, the structures occurring
naturally in food and commercial prepara-
tions may actually be provitamins that have to
be converted to the biochemically active form.
Unless specified differently, the information
in the following summaries is found in the Di-
etary Reference Intakes, published by the Insti-
tute of Medicine (5-8).
3.1 Retinol (Vitamin A) Family (9)
Early work on this vitamin was confusing be-
cause similar outcomes were seen with inges-
tion of "yellow" vegetables and colorless fish
liver oils. It finally was shown that carotene
(the yellow pigment) extracts from vegetables
were converted to colorless retinal. Because
the retinoids are discussed in considerably
more detail elsewhere, this chapter presents
only a cursory overview of their biochemical
functions. .
3.1.1 Chemistry. The commercial form of
vitamin A is all-trans retinol, usually fomu-
lated as the acetate or palmitate ester. The
active forms are the two oxidation products
(Fig. 8.1, (1) retinal, which is a structural
component of the visual pigment rhodopsin,
and (2) retinoic acid, which is required for cell
differentiation. There are specific nuclear re-
ceptors for retinoic acid. Although the vitamin
is marketed in the all-trans form, retinal and
retinoic acid are present, in vivo, in cis forms.
There are also commercial forms related to the
retinoic acid structure that have cis
stereochemistry.
Carotenes are promoted commercially for
their antioxidant activity rather than as a
source of the retinol group. Nevertheless, the
carotenes are the source of the vitamin in yel-
low vegetables. There are many carotenes, of
which three are shown in Fig. 8.2. Only p-car-
otene is symmetrical (note the dashed line)

Retinol (commercial form)
I
I
lo]
Retinal (vision)
Retinoic acid (cell differentiation)
Figure 8.1. Retinol chemistry.
and theoretically will produce two equivalents
fretinalafter the enzyme-catalyzed oxidative
cleavage.In reality, more recent studies indi-
a t e that vitamin A activity is six times the
Pitamin A activity derived from p-carotene.
b e problemis one of capacity in the intestinal
bucosa cell to cleave the carotenes. Further,
appears to be regulation of the cleavage
Figure 8.2. Carotene chemistry.
of p-carotene. As the stores in the liver reach
capacity, there is less conversion of p-carotene
being oxidized to retinal. This is one of the
reasons that p-carotene nutritional supple-
ments enter the body intact. Further, the bio-
availability of p-carotene is significantly lower
than that of retinol(10).
3.1.2 Uptake and Metabolism (Fig. 8.3).
Both retinol esters, whether from animal
tissues or a vitamin supplement, and p-car-
otene must be incorporated into mixed mi-
celles along with other lipid material. The
retinol esters are hydrolyzed by intestinal
esterases. Both retinol and p-carotene are
then absorbed into the mucosa cell, where
p-carotene is oxidatively cleaved to retinal
and then reduced to retinol. The retinol, in-
dependent of the source, follows the same
steps seen with 2-monoglyceride from tri-
glyceride digestion. The retinol is reesteri-
fied, usually with palmitic acid, and attached
to the chylomicrons along with the other di-
etary lipids. The chylomicrons first enter the
lymph and then move to the circulatory sys-
tem. The triglycerides are removed from the
chylomicrons and deposited in the adipose
and skeletal muscle cells, leaving chylomi-
CrOn remnants, which are transported to the
.
liver where the esterified vitamin is stored
(11).Transportation from the liver to tis-
sues where required is done on specific reti-
nol-binding proteins. Humans consuming a
balanced diet store several months of retinol
esters in their livers.

oxidation mucosa cell
Pcarotenemucosa cell * retinal \ reductase
Retinol esters
(food or vitamin supplements)
retinal & retinoic acid (1 ) esterase liver
transported on Retinal * (2) oxidation storage
Binding Protein (RBP)
Figure 8.3. Uptake and metabolism of retinol esters and p-carotene.
3.1.3 Biochemical Functions and Deficiency.
Two retinoids, retinoic acid and retinal, ap-
pear to have most of the biochemical functions
attributed to vitamin A. Retinoic acid is re-
quired for cell differentiation and is the ligand
for two families of nuclear receptors,
and RXR,,B,,. These receptors are part of a
family of superreceptors that include the ste-
roid hormones and cholecalciferol. Vitamin A
deficiency can lead to a variety of symptoms
depending on the age of the deficient person.
The most serious syndrome is keratomalacia,
which results in desiccation, ulceration, and
xerophthalmia of the cornea and conjunctiva.
It is one of the leading causes of blindness in
infants and children.
Retinoic acid is required for the development
of goblet mucous cells. A deficiency results in
basal cell proliferation with increased keratini-
zation of the epithelial structures. Mucus is one
of the essential physical barriers (part of innate
immunity) that prevents pathogens from enter-
ing the body. Therefore, a retinol deficiency in-
creases the risk of infection.
The aldehyde form, retinal, is an essential
component of the visual pigment found in the
rods of the eye. A very brief outline of the rho-
dopsin cycle is shown in Fig. 8.4. Retinol is
transported from the liver to the eye, where it
is converted to 11-cis-retinal. In the rod, the
aldehyde forms an enamine (Schiff's base)
with a lysine on opsin forming rhodopsin (Fig.
8.5). In the presence of light, trans-retinal
forms with cleavage of the enamine, sending a
nerve impulse to the brain along the optic
Vitamins
A
retino1
reesterify
mucosa cell - retinol palmitate
/;ntestinal
esterases
TGs
4
chylomicron chylomicron
remnent
nerve. In the dark, 11-cis-retinol re-forms fol-
lowed by oxidation to 11-cis-retinal and the
cycle repeats.
A deficiency causes night blindness, which
is considered an early symptom of retinol de-
ficiency. Night blindness refers to decreased
ability to see in very dim light because there is
an inadequate amount of retinal in the eye to
fully "stock" the rods with functional rhodop-
sin. There is some evidence that as retinol lev-
els in the liver decrease, the equilibrium fa-
vors the movement of retinol from the eye
back to the liver.
3.1.4 Dosage Forms. Retinol is unstable. It
easily dehydrates (Fig. 8.6), forming a stable
carbonium ion. Therefore, the two most com-
mon forms for both oral and p a r e n t e d ad-
ministration are the acetate or palmitate es-
ters. With the extensive conjugation retinol is
light sensitive and subject to oxidation. There-
fore, the vitamin formulator must protect the
product from light and air.
3.1.5 Hypervitamininosis A. In high doses,
retinol can be toxic. Acute poisoning is rare
and dependent on the dosage form. Nausea
and vomiting are the most common symp-
toms. Most rapidly absorbed are the "clear"
emulsions (usually formulated with a Tween
or other surfactant). Next in order are the
standard emulsions, usually produced from
fish liver oils. The most slowly absorbed are
the dry tablet formulations or an oil solution
in a capsule. Chronic hypervitaminosis is
 Rhodopsin
(visual pigment)

/
/ (Dark)
Changes in the conformation
of the Rhodopsin complex
Nerve impulse
to the brain
Sight

11-&-Retinal + Opsin trans-Retinal + Opsin

trans-Retinol
(transported to the eye
from the liver on a retinol
binding protein)

Liver stores of
retinol esters

Figure 8.4. Outline of the rhodopsin cycle.

re common and is more commonly seen seem to be no other symptoms. The skin col-
n people consume fish liver oil concen- oration slowly disappears when carotene in-
es. The symptoms are nondescript and in- take stops. A commercial form of carotene is
de fatigue, malaise, lethargy, abdominal indicated for the photosensitivity seen in
comfort, boneljoint pain, severe and throb- erythropoietic porphyria. Carotene capsules
g headache, insomnia, restlessness, dry are also sold with the claim that a person can
d scaly skin, loss of body hair, brittle nails, have a tanned appearance without the need of
tipation, and irregular menses, symptoms UV radiation. Patients who drink large
ight make users conclude that they are amounts of carrot juice sometimes show signs
in A deficient. Depending on the health of hypercarotenosis.
person's liver, there is risk of developing
hosis. There is a daily Tolerable Upper In- 3.1.7 Dietary Reference Intakes (as Retinoic
e Level (UL) of 3000 pg for this vitamin. Acid Equivalents).
e UL to RDA ratio is narrow (3-51, relative
that of most vitamins. This is somewhat A1
mparable to vitamins D. Infants (1-12 months) 400-500 pglday
EAR
3.1.6 Hypercarotenosis. This occurs from Children (1-8 years) 210-275 pglday
sive doses of carotene that exceed the ca- Boys (9-18 years) 445-630 pglday
ty of the mucosa cells to cleave the mole- Girls (9-18 years) 420485 pglday
to retinal derivatives. The excess carotene Men (19-70+ years) 625 pglday
omes deposited in the body tissues. Except Women (19-70-t years) 500 &day
the yellow or bronze-orange skin, there Lactating 885-900 pglday
 Vitamins

RDA
Children (1-8 years) 300-400 pglday
Boys (9-18 years) 600-900 pglday
Girls (9-18 years) 600-700 pglday
Men 900 pglday
Women 700 pglday
Pregnant 750-770 pglday
Lactating 1200-1300 pg/
day
UL

1'
Opsin
3000 &day for all adults including
pregnant women. There is some concern of
teratogenic effects based on the experience
of the retinoids used in therapy.

3.1.8 Pharmacologically Active Retinoids.

Because retinol deficiency results in keratini-
zation of epithelial tissue, at one time retinol
was recommended for skin conditions includ-
ing acne. There is no clinical evidence that ret-
in01is effective for skin conditions. Now that it
is realized that the active form is retinoic acid,
the focus has been on developing pharmaco-
logically active compounds based on this
structure. These are divided into treatment of
three groups: (1) acne, (2) the autoimmune
5
disease psoriasis, and (3) malignancies.
Rhodopsin 3.1.8.1 Retinoid and Retinoid-like Drugs
Used in the Treatment of Acne (Fig. 8.7) (12).
Figure 8.5. Rhodopsin. The first product introduced was tretinoin,
which is a topical all-trans retinoic acid. Its
effectiveness may not be related to any direct
retinoid activity, but attributed by its produc-
ing a complex response related to increasing

R = acetate or palmitate

Figure 8.6. Commercial retinol esters.

 the turnover of follicular epithelial cells. The
result is decreased cohesiveness of follicular
epithelial cells.
Tretinoin is also used as an antiwrinkle
cream. There is disagreement on the mecha-
nism and there may be more than one. Some
Tretinoin (Retin
Retinoic acid improvement may be attributable to the irri-
tation the drug causes. There is an increase in
epidermal cell turnover, shedding older cells
and thickening the skin. Also the drug may
combine with epidermal retinoic acid recep-
tors, thereby decreasing keratin production.
Keratin can contribute to skin wrinkling (13).
O""\OH Isotretinoin, 13-cis-retinoic acid, is very ef-
fective at treating severe forms of acne. It also
lsotretinoin ( ~ c c u t a n e ~ ~ )
13-cis-Retinoic acid is very teratogenic. There are elaborate proce-
dures involving the prescribing physician and
dispensing pharmacist before a female patient
can receive the drug. There is also some con-
cern that the sperm of men using the drug
'OH
might be affected.
Although used topically, the nonretinoid,
adapalene, does bind to the retinoic acid nu-
clear receptor and does affect cell differentia-
tion, keratinization, and inflammatory re-
sponses.
3,1.8.2 Drug Based on the Retinoid Struc-
ture Used to Treat Psoriasis (Fig. 8.8). Etrinate
is the ethyl ester of acitretin and is active after
Adapalene (Dierin el^^) hydrolysis to the acidic drug. The "terminal"
half-life after 6 months of etrinate therapy is
re 8.7, Retinoid and retinoid-like drugs used 120 days. In contrast, the "terminal" half-life
in the treatment of acne. of acitretin is only 33-96 h. Both drugs are
3
*'

a, 1

Etretinate (TegisonTM);R = CH;

Acitretin (soriateneTM);
R =H

V Figure 8.8. Drug based on the retinoid

Tazarotene Gel structure used to treat psoriasis.

Alitretinoin (panretinTM)
9-cis-Retinoic Acid
0 that rickets was not found in sunny climates,
Bexarotene (Targretin TM)
Figure 8.9. Retinoids used in the treatment of
malignancies.
teratogenic and require elaborate warnings
before being prescribed and dispensed. The
third drug, tazarotene gel, is administered
topically and is indicated for both acne and
psoriasis. Although there appears to be mini-
mal absorption if used over a limited skin area,
there is some absorption, with retention by
the body for up to 3 months. It also can cause
fetal damage and cannot be used by pregnant
women or women who may become pregnant.
3.1.8.3 Retinoids Used in the Treatment of
Malignancies (Fig. 8.9). Retinoic acid has been
evaluated as a possible treatment for malig-
nancies. This is based on the fact that it is
required for proper cell differentiation and
products based on the retinoid structure are
teratogenic. All-trans retinoic acid is used in
combination with other chemotherapeutic
agents for the treatment of acute promyelo-
cytic leukemia (14). It does not kill the malig-
nant cell, but seems to facilitate the cell's
differentiation into a nonproliferating myelo-
cyte. Alitretinoin, 9-cis-retinoic acid, is indi-
cated for Kaposi's sarcoma and is adminis-
tered topically. It binds to all six retinoic acid
receptors.
Bexarotene is most selective for the three
RXR,,p,, receptors. It also binds several other
nuclear receptors including the RAR, vitamin
Vitamins
D, thyroid, and peroxisome proliferator acti-
vator receptors, which probably explains its
numerous adverse reactions. The drug is indi-
cated for cutaneous T-cell lymphoma and is
available as capsules and a topical gel.
3.2 Vitamin D Family (15)
Rickets was first reported in the mid-seven-
teenth century. It could be lethal, but bone
deformation was more common. In the United
States, rickets continued to be a problem into
the 1930s until vitamin D-fortified milk be-
came common. Older adults show bowlegs
characteristic of childhood rickets. There were
many attempts at giving calcium andlor phos-
phorous supplements. Finally, it was realized
and populations whose main source of protein
was fish did not have rickets. In 1924, Profes-
sor Henry Steenbock, University of Wiscon-
sin, demonstrated that irradiation of foods, in-
cluding milk, produced food that was
antirachitic (16, 17).
When there is adequate sunlight, no di-
etary source of the vitamin is required. In-
deed, an argument can be made that the cal-
ciferols are not normal components of the diet.
In the United States. it is added to milk. other
dairy products, and dairy substitutes. Fish is
about the only natural food source. ~holecal-
ciferol is produced in the body from endog-
enously synthesized 7-dehydrocholecalciferol
(Fig. 8.10). Consistent with a hormone model,
excess amounts of cholecalciferol can result in
excess calcium uptake from the intestinal
tract, leading to calcification of soft tissues.
3.2.1 Chemistry. There are two forms ofvi-
tamin D, and both are considered biologically
equivalent. Irradiation of the major plant ste-
rol, ergosterol, produces ergocalciferol, also
known as vitamin D, (Fig. 8.11). Because they
are photochemical reactions and in contrast to
enzyme-catalyzed biochemical reactions, the
formation of cholecalciferol is not clean. Expo-
sure of human skin to sunlight of 295-300 nm
converts 7-dehydrocholesterol to previtamin
D,. The isomerization to cholecalciferol (vita-
min D,) is heat catalyzed. Continuous expo-
sure to ultraviolet radiation from the sun re-
sults in the reversible formation of lumisterol

Cholecalciferol (Vitamin D3)
Figure 8.10. Photochemical formation of cholecalciferol (vitamin D3).
tachysterol (18, 19). Once the B-ring of
two steroids has been cleaved, the prod-
should no longer be referred to as ste-
roidal vitamins.
the 7-dehydrocholesterol in the skin. In
ntext D vitamins, when administered in
ements, could be considered replace-
therapy. When photochemically synthe-
specific binding protein formed in the skin
7-Dehydrocholesterol
Tachysterol
shown in Fig. 8.10 toward the desired chole-
calciferol. Dependency on the binding protein
for transport from the skin also provides a
rproduction of the hor-
mone and possible hypercalcemia.
When taken orally, it follows the same route
mucosa cells. It is then
the chylomicron rem-
ogenously produced
cholecalciferol from sunlight, it is hydroxylated
to 25- OH-cholecalciferol.
The 25-hydroxylated product is then trans-
ported to the kidney where it is hydroxylated a
-1 a-hydroxylase,
forming the active 1,25-dihydroxycholecalcif-
ter is transported
e it attaches to a
the mucosa cell

Ergosterol
I
uv
Ergocalciferol (Vitamin D2)
Figure 8.11. Formation of ergocalciferol from
ergosterol.
to synthesize a calcium transport protein. The
final 1,25(OH),D3 product can be considered a
kidney hormone that regulates calcium in-
take. Finally, 1,25(OH),D3 is oxidized to inac-
tive calcitroic acid that is excreted through the
kidney. The 24,24(OH),D3 metabolite may be
part of the degradation process for 25(OH)D3
that is not transported to the kidney, but it
also can elevate serum calcium levels (16). Pa-
tients with kidney failure can experience vita-
min D-resistant rickets. Because they cannot
carry out the final hydroxylation step,
1,25(OH),D3 (calcitriol)is prescribed for their
hormone replacement therapy.
3.2.3 Biochemical Function. Calciferol func-
tion is complex and, with the exception of cal-
cium transport from the intestinal tract, is
poorly understood. Specific vitamin D recep-
tors (VDRs) are found in 30 different tissues
including bone, intestine, prostate, hemato-
Vitamins
poietic cells, and skin (20). Like the retinoic
acid receptors, the vitamin D receptor is a nu-
clear receptor belonging to the steroid hor-
mone superfamily of nuclear receptors, which
includes receptors for estrogen, glucocorti-
coids, thyroid hormones, and retinoic acid.
There are at least 50 genes that respond to
hydroxylated calciferols regulating calcium
release and uptake and cell division.
3.2.3.1 Calcium Regulation. There are at
least three hormones that regulate calcium
metabolism, parathyroid (PTH), calcitonin,
and 1,25(OH),. Bone is the principal calcium
reservoir and is a dynamic tissue, with cal-
cium being released and deposited. New
calcium comes from our diet, and excess serum
calcium is excreted through the kidneys. In
response to low serum calcium levels, PTH
stimulates the hydroxylation of 25(OH)D,,
leading to formation of calcium transport pro-
tein and activation of osteoclast cells required
to release calcium from bone. PTH also inhib-
its calcium excretion by the kidney. In con-
trast, calcitonin (produced in the thyroid
gland) acts when serum calcium levels are
high. It promotes the deposition of calcium
into bone by osteoblast cells and excretion of
calcium by the kidney.
Rickets in infants and children and ostea-
malacia, and possibly osteoporosis, in adults is
caused by a deficiency of D vitamins. Today
most deficiencies are caused by a lack of sun-
light or restricted diet. The lack of sunlight
may be caused by living in northern latitudes.
The latter also can be affected by the amount
of skin pigmentation (21-23). A strict vegetar-
ian diet lacking cholecalciferol-fortified dairy
products or fatty fish, particularly in children,
may also result in rachitic lesions in the bone
(24,251. Mechanistically, rickets and osteoma-
lacia are similar and are characterized by bone
softening. Normal bone growth and mainte-
nance require that the osteoblast cells lay cal-
cium hydroxyapatite onto a cartilage matrix.
A deficiency of D vitamins results in a lack of
mixed calcium salt available to the osteoblast
cells. In infants and children, the cartilage
continues to grow. Cartilage, being soft, can-
not support the child's weight, leading to the
typical bowlegs seen in a rachitic child. An
 3 Vitamins
I
HO
PP
1,25(OH)2D3
(Calcitrol)
(Kidney)
HO"' OcH2
25(OH)D3
1
I
!
6 (Intestine)
HO D3
Calcitroic acid
Figure 8.12.
Metabc~ l i s m
adult also will have bone deformations, partic-
ularly in the pelvic area, because the bones
cannot support the heavy upper torso.
Osteoporosis is a Merent disease. It can be
thought of as osteoclast cells removing calcium
more quickly that osteoblast cells can lay cal-
eium down. The result is porous, brittle bones
that break easily. At one time calcitonin some-
times was mescribed to decrease the release of
osteoclast cells. In addi-
lates and irnpact exercise,
i n s is cur~ e n t l yrecom-
:ium being released from
- the ki.hey.
)ugh
3.2.3.2 Vitamin D Analogs Used in Chronic
la1 Failure. Because 1.25(OH),DQ
.a u is Dro-
* calcitriol). It is not clear why doxercalciferol is
renal fail1Ire leads to a
none a n d .vitamin D-re-
t rickets. The solution is prescribing
hetic 1,25(OH),D3 (generic name: calcit-
I). Nevertheless, these patients can experi-
2 hyperparathyroidism caused by the para-
oid gland attempting to compensate for
(Kidney)
*
\
(Liver) (Liver & kidney)
Variety of hydroxylated
and carboxylic acid products
(Ref 17)
o f D vitamins.
the lack of 1,25(OH),D3. The result is in- .
creased osteoclast activity, leading to loss
of calcium from the patient's bones, further
resulting in hypercalcemia and either osteo-
malacia or osteoporosis. To overcome these
complications,two synthetic ergocalciferol an-
alogs (Fig. 8.13) are indicated for secondary
hyperparathyroidism associated with chronic
renal failure. Note that both compounds con-
tain the hydroxy group at position 1, the posi-
tion at which the kidney carries out its hy-
droxylation to produce the 1,3,25-trihydroxy
product. Doxercalciferol is 1,3-dihydroxy-er-
gocalciferol and, in the liver, is hydroxylated to
active 1,25(OH),D2 (the ergocalciferol analog of
more selective than calcitriol. In contrast, pari-
calcitol is the 19-normethyl analog of
1,3,25(OH),D2 produced from ergocalciferol
and requires no further hydroxylation reactions.
3.2.3.3 Cell Division. The role of 1,25(OH),
D vitamins in regulating cell division is under
active investigation. There are many reports

Doxercalciferol (HectoralTM)
1,3-dihydroxy-ergocalciferol
Paricalcitol (2emplarTM)
19-nor-1,3,25-trihydroxy-ergocalciferol
Figure 8.13. Ergocalciferol analogs indicated for
chronic renal failure.
that indicate that vitamin D has a cancer-pre-
ventive role. Populations living in areas with
higher exposure to sunlight have lower inci-
dence of prostate, breast, and colon cancers (26).
In the intestine, the vitamin D receptor
also functions as a receptor for lithocholic
acid, a bile acid produced by the action of in-
testinal bacteria on endogenously produced
chenocholic acid. Lithocholic acid is hepato-
toxic and may be a carcinogen. In the intes-
tine, vitamin D receptors, activated by vita-
min D and possibly lithocholic acid, induce
both liver and intestine cytochrome P450 3A
oxidase (CYP 3A). This enzyme metabolizes
lithocholic acid to inactive hydroxylated
forms. The process of inducing CYP 3A might
Vitamins
OH
HO'"'
Calcipotriene (I3ovenexTM)
Figure 8.14. Calciferol analog indicated for psoriasis.
explain how vitamin D may exert its protec-
tive effect against cancer of the colon (27).
In contrast with other vitamins that are
associated with reducing cancer risk and have
minimal adverse reactions in high doses, such
as a-tocopherol and ascorbic acid, taking
larger doses of D vitamins can lead to clinically
significant hypercalcemia. Therefore, the
challenge is to develop compounds that not
only are selective for receptors involved with
controlling cell division but also will activate
calcium transport leading to hypercalcemia.
The initial analog on the market is calcipot-
riene, which is indicated for psoriasis, a nbn-
malignant proliferation of cells (Fig. 8.14). Its
use is limited to topical application. When ad-
ministered internally, hypercalcemia can re-
sult. Attempts have been made at pulse dosing
of 1,25(OH),D3, to maximize inhibition of cell
division with minimal calcemic activity (28).
Most of the calciferol analogs are based on
modifications of the 17-alkyl side-chain.
Modifications of the A-ring include 19-nor
methylene and 3-nor hydroxy analogs with
and without the 1-hydroxy moiety. The la-
hydroxy-24-ethyl cholecalciferol analog (Fig.
8.15) was less calcemic in mice and inhibited
the development of preneoplastic lesions in
mammary glands (29). Unsaturation at posi-
tion 16 (Fig. 8.16) provides modest antiprolif-
erative effects on prostate cancer cells with
little hypercalcemia (30). A series of 20-cyclo-
propyl-cholecalciferol analogs (Figs. 8.17 and
8.18) showed good activity against human
prostate, breast, and myeloid leukemia cell
 379

ment. It is stabilized with antioxidants and

protective coatings.

3.2.5 Hypervitaminosis D. Think of a vita-

min D overdose in the same way as an over-
supply of any hormone. The role of the
hormone is exaggerated or magnified. Hy-
pervitaminosis D causes increased absorp-
tion of calcium and phosphorous (P follows
Ca), leading to calcification of the tissues,
vomiting, kidney damage, and so forth. It
can be the most serious of the hypervitamin-
1-(OH)-24-ethyl-cholecalciferol oses. The reports that D vitamins inhibit
proliferation of tissue and may protect
Figure 8.15. "la-HydroetaminD,." against certain cancers could cause the pub-
lic to overdose with this vitamin. The calcif-
lines (31). The most potent in this series were erols have ~ ~ l upper ~ ~~~~k~
~ ~ ~~~~l~
b l ~
the 19-nor methylene analogs, independent of (UL) with UL to RDA ratios of 5 to 10.
whether the side-chain had ethylene or acety-
lene unsaturation. Modification of the side 3.2.6 Dietary Reference Intakes. There is
chain with a 25 keto or oxime (R, = 0 or NOH, no RDA for this vitamin. It would be difficult to
res~ectively),with or without an additional derive a RJJA for a population because a signifi-
hydrogen or methyl (R,= Or CH3, respec- -t percentage will receive adequate amounts
tively), produced analogs as antiproliferative of the vitamin from sunlightbased on the area of
in uitro 1,25(0H)2D3 (Fig. 8.19). The the country where they live. On the other hand,
mimes were less calcemic (32). the diverse population of the United States
3.2.4 Dosage Forms. The commercial means that people of color may need more of the
odu& are produced by irradiation of 7-de- vitamin from fortified milk relative to those
lesterol or ergosterol under con- whose ancestors came from Northern Europe.
lled conditions. The final yield is about
.
ough more stable than vitamin A, D Infants (0-12 months) 5 pg (200 IU)/day
ns are sensitive to oxygen and tend to Children (1-8 years) 5 pg (200 IU)/day
inactive isomers in the pres- Boys (9-18 years) 5 pg (200 IU)/day
ace metals, which can cause prob- Girls (9-18 years) 5 pg (200 IUYday
lating a vitamin/mineral supple- Men (19-50 years) 5 pg (200 IU)/day
Women (19-50 years) 5 pg (200 IU)/day
Men (51-70 years) 10 pg (400 IU)/day
Women (51-70 years) 10 pg (400 1U)lday
Men (70+ years) 15 pg (600 IU)/day
Women (70+ years) 15 pg (600 IU)/day
Pregnancy 5 pg (200 IU)/day
Lactation 5 pg (200 IU)/day
UL
Infants 25 pg (1000 IU)/day
Children (1-18 years) 50 pg (2000 IU)/day
Adults (over 19 years) 50 pg (2000 IU)/day
Pregnancy 50 pg (2000 IU)/day
Lactation 50 pg (2000 IU)/day
1 ,25-(OH)2-16-ene
3.2.7 Drug Interactions. Phenobarbital and
Figure 8.16. 16-ene-23-yne D, analogs. possibly other anticonvulsants used in epi-

Figure 8.17. 23-yne-20-cyclopropylD, analogs.
lepsy induce liver hydroxylation, leading to
subsequent formation of the inactive end
products. As long as the epileptic child re-
ceives a normal amount of fortified milk, there
is no problem with this interaction.
3.3 Vitamin E Family (Tocopherols
and Tocotrienols) (33)
This vitamin group was discovered in rat feed-
ing experiments, which resulted in these ani-
Vitamins
mals not being able to produce offspring. These
same rats seemed normal in all other respects
including physical growth. The condition could
be corrected by addition of lettuce, whole wheat,
and cereal grains, with the best source being
wheat germ followed by vegetable oils. Think of
the tocopherols and tocotrienols (Table 8.3) as
nature's antioxidants. Most of the vitamin activ-
ity is found in a-tocopherol. "Tocopherol"
means child-bearing alcohol.
l Vitamins
'igure8.18. 23-E-ene-20-cyclopropyl D, analogs.
3.3.1 Chemistry. The vitamin E family
mists of tocopherols and tocotrienols. There
as been considerable debate as to whether
ie RRR isomer is the only biologically active
~ r mof the vitamin. The vitamin of commerce
I a-tocopherol, either as a racemic synthetic
k p e 8.19. Keto and oxime cholecalciferol
mixture or the natural RRR-a-tocopherol. All
of the tocopherols found in food have the R
configuration at position 2. The synthetic
form of the vitamin is a mixture of all eight
stereo isomers (now referred to as rac-a-to-
copherol rather than d,l-a-tocopherol). Be-
cause there is both general and stereospecific
antioxidant activity, the RDA tables state that
the activity ratio of RRR-a-tocopherol to rac-
a-tocopherol is 1 to 1.36 (Table 8.4) (34). All
isomers are antioxidants and probably do pro-
vide general antioxidant protection internally
or in vivo. On the other hand, evidence points
to the RRR isomer as being specific for a vari-
ety of reductase systems, possibly involving
selenium (Se) and glutathione (GSHIGSSG).
The "Relative Biological Activity" in Table 8.3
is based on a rat resorptionlgestation assay, as
are the conversion factors in Table 8.4.
3.3.2 Uptake and Metabolism. Like the
retinol esters, tocopherol, both esterified and
nonesterified, requires bile salts to become
part of the mixed micelles containing the di-
etary lipids. They are absorbed into the muco-
sal cell by passive diffusion. The tocopherols
follow the dietary lipids onto the chylomi-
crons. Because the latter first enter the lym-
phatic system before the circulatory system,
the tocopherols do not go directly to the liver.
Instead, the chylomicrons are distributed
throughout the body, and some of the toco-
pherols enter the adipose tissue with the fatty
acids that were also being transported on the
chylomicrons. The chylomicron remnants fi-
nally reach the liver. The remaining tocoph-
erols leave the liver on the very low density
lipoproteins (VLDL), which become the low
density lipoproteins (LDL). In other words, to-
copherols will be found wherever there is a sig-
nificant amount of lipid material, including the
high density lipoproteins (HDL). There is no
specific organ where the tocopherols are stored.
Consistent with antioxidant model, the RDA is
based on the polyunsaturated fatty acid (PUF'A)
consumption. The implication is that, if in-
creased PUF'A intake is recommended, the RDA
for tocopherol should be increased.
There is increasing evidence that the RRR
stereoisomer is preferentially transferred in
the liver onto the lipid transport proteins, but
it is not absolute. This could explain why
382 Vitamins

Table 8.3 Relative Biological Activities of Tocopherols and Tocotrienolsa

Tocopherols Relative biological activieyb

Tocotrienols R1 R2 Relative Biological Activityb

0.3
0.05
Inactive
Inactive
"Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids, Food and Nutrition Board, Institute of
Medicine, National Academy Press, 2000, p. 193.
'Rat fetal reabsorption assay.

Table 8.4 Factors for Converting International Units of Vitamin E to a-Tocopherol (mg)
to Meet Recommended Intake"
Molar a-Tocopherol
USP conversion factors
b conversion conversion
factors factors
UUfmg) (mg/IU) (~movIU) (mg/IU)
Synthetic vitamin E and estersc
d,l-a-Tocopherolacetate 1.00 1.00 2.12 0.45
d,l-a-Tocopherolsuccinate 0.89 1.12 2.12 0.45
d,l-a-Tocopherol 1.10 0.91 2.12 0.45
Natural vitamin E and estersd
d-a-Tocopherol Acetate 1.36 0.74 1.56 0.67
d-a-Tocopherol Succinate 1.21 0.83 1.56 0.67
d-a-Tocopherol 1.49 0.67 1.56 0.67
"Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium and Carotenoids, Food and Nutrition Board, Institute of
Medicine, National Academy Press, 2000, p. 192.
*The United States Pharmacopeia (USP) has defined one IU as 1 mg of all racemic a-tocopherol acetate based on a 1940s
rat fetal resorption assay.
"Synthetic vitamin E supplements labeled as d,l-a-tocopherol can consist of all eight possible isomers (RRR-,RSR-, RRS-,
RSS-, SSS-,SRS-, SRR-, and SSR-).
dd-wTocopheroI refers to the RRR-isomer, the only one found naturally in foods, and the other two R stereoisomers
(RRS-, RSR-, and RSS).

Chromanoxyl radical Chromanol methide radical
Figure 8.20. Resonance-stabilized tocopherol radicals.
RRR-a-tocopherol is more active in vivo, but
the other isomers are also active.
3.3.3 Biochemical Function. The best way
to describe tocopherol's role is that of a lipid-
soluble antioxidant. It protects unsaturated lip-
ids from oxygen-induced peroxide formation.
Thereis evidence for both free-radical one-elec-
tron chemistry (Fig. 8.20) and two-electron qui-
none-hydroquinone chemistry (Fig. 8.21) (35).
The oxidizedlreduced glutathione system may
be part of the system that regenerates reduced
a-tocopherol. At one time it was thought that
the preference for the 2-E1 stereoisomers indi-
cated that the vitamin was part of a biochemical
oxidationlreduction system, possibly as a coen-
zyme. So far that role for a-tocopherol has not
been found. The current evidence points to the
Reduced Tocopherol (hydroquinone)
[HI [Ol
Oxidized Tocopherol (quinone)
CH3
-
CH3
Chromanoneda radical
hepatic tocopherol transfer protein's preference
for the RRR stereoisomer as the explanation for
the partial steric preference.
3.3.4 Deficiencies. The current model for
the cause of vitamin E deficiencies points to
malabsorption of lipids. Thus, there may be
malabsorption of other lipid-soluble vitamins.
Little is known regarding the pharmacokinet-
ics of the tocopherols. Part of the reason for
this may be attributed to lack of a specific stor-
age organ for the vitamin.
Correlating human medical conditions
with the biochemical role of the tocopherols is
difficult because of the lack of correlations be-
tween deficiency diseases seen in animals rel-
ative to what is seen in humans. Deficiency
diseases seen in animals include reversible re-
Figure 8.21. Tocopherol oxida-
tiontreduction.
 Vitamins

Figure 8.22. a-Tocopherol dos-

age forms. R = acetate, hemisuccinate (sodium salt)

productive failure in female rats; irreversible commercially (Fig. 8.22), the oil-soluble ace-
degeneration of rat testicular tissue leading to tate and water-soluble hemisuccinate. The lat-
male sterility; nutritional muscular dystro- ter is commonly found in dry dosage forms
phies in monkeys, rabbits, guinea pigs, lambs, requiring a free-ffowing powder. Oxidation to
calves, turkeys, and chicks; and an anemia in the quinone form is blocked by esterifying the
monkeys. Vitamin E does not treat human free phenolic hydroquinone.
muscular dystrophy nor the various causes
3.3.7 Dietary Reference Intakes (based on
preventing a couple from conceiving a child or
d-a-Tocopherol).
inability of pregnant women to go to full term.
What is seen in humans is a partially revers- A1
ible set of neurological problems and hemo- Infants (0-12 months)
lytic anemia in premature infants. Because of EAR
poor placental transfer, newborns have little Children (1-8 years) 5-6 mglday
of the vitamin. Human milk contains 2-5 Boys (9-18 years) 9-12 mglday
mg/L; cow's milk contains less. Girls (9-18 years) 9-12 mglday
There are numerous studies evaluating the Men (19-50 years) 12 mglday
possible role of tocopherols in preventing Women (19-50 years) 12 mg/day
and/or treating cardiovascular disease, malig- Men (51-70+ years) 12 mg/day
nancies, diabetes mellitus, cataracts, immune Women (51-70+ years) 12 mg/day
function, and Alzheimer's Disease. In all of Pregnancy 12 mglday
these conditions, there is evidence of free-rad- Lactation 16 mglday '

ical formation or general oxidation mecha- RDA

nisms as part of the disease process. The evi- Children (1-8years) 6-7 mg/day
dence for taking supplements in addition to Boys (9-18 years) 11-15 mglday
proper diet is mixed (36, 37). Girls (9-19 years) 11-15 mglday
Men (19-50 years) 15 mglday
Women (19-50 years) 15 mglday
3.3.5 Hypervitaminosis E. This is a rela- 15 mglday
Men (51-70+ years)
tively safe vitamin. Toxicities have been re- Women (51-70+ years) 15 mglday
ported involving chronic administration of Pregnancy 15 mglday
300-1200 mg per day. The symptoms can be Lactation 19 mg/day
very serious and include thrombophlebitis, UL
pulmonary embolism, hypertension, breast Infants Not established
development in men and children, severe fa- (Do not give
tigue, and nonmalignant breast tumors. Nev- supplements;
ertheless, the UL to RDA ratio is about 66 to 1 use only food
for adults, making it a very safe vitamin. and formula for
sources.)
3.3.6 Dosage Forms. The tocopherols, be- Children 200 mglday (1-3
ing antioxidants, are very sensitive to oxygen. years) up to 600
Sensitivity to UV light is another problem. mglday
There are two provitamin esters that are used (9-13 years)
3 Vitamins
Phytonadione (Vitamin K1;phylloquinone)
I Removal of the side chain
Menadione
I Addition of the geranylgeranyl chai~
Vitamin K2 (n = 4; menoquinone-4)
Adolescents 800 mg/day
Adults (19+ years) 1000 mg/day
Pregnancy 800-1000 mglday
Lactation 800-1000 mg/day
.4 Vitamin K Family (38)
[itaminK was discovered by accident by Dan-
sh scientists who, using a special fat-free diet
lesigned to determine whether chickens syn-
hesize cholesterol, observed that the animals
/evelopeda hemorrhagic condition character-
eed by a prolonged clotting time. The condi-
ion could be cured by an organic factor found
0 fresh cabbage, ether extract of alfalfa, pu-
refied fish meal, cereals, or hog livers. It was
wed Vitamin K for koagulation vitamin.
Bhis may be the only vitamin that humans
Figure 8.23. Formation of menoquinone form
of vitamin K.
receive in significant amounts from their in-
testinal bacterial, and there is some question
regarding this commonly held assumption.
Because of this source, it has been very diffi-
cult to establish a recommended daily allow-
ance. An estimated safe intake was estab-
lished for this vitamin for the first time with
the 1989 RDA tables. With the release of the
Dietary Reference Intakes, there is an ade-
quate intake, but no RDA.
3.4.1 Chemistry. There are two series for
this vitamin (Fig. 8.23). The vitamin K, series
is mostly obtained from green plants, whereas
the K, series is the product of bacteria. The
active vitamin is in the K, series. Menadione
has sometimes been referred to as vitamin K,.

aCH3
OH
R
Several
steps
O2
Vitamin K2H2
Thioredoxin
R X-(S)2 X-(SH)z
0 0
Vitamin K2(20)
Figure 8.24. Outline of vitamin K in carboxylation of glutamic acid.
The common commercial form is called phy-
tonadione in the United States Pharmacopeia
and phylloquinone by Chemical Abstracts.
3.4.2 Vitamin K Uptake and Metabo-
lism. Dietary vitamin K, and the pharmaceu-
tical form, phytonadione or vitamin K,,,,,
must be converted to the K, series known as
menoquinones. The most common of these is
menoquinone-4 or K,,,,,. This conversion to
the K, series occurs in the liver and possibly
the intestinal flora. It involves removing the
phytyl chain producing the intermediate men-
adione. Menadione sometimes is prescribed
when there is impaired uptake of lipids from
the intestine. There is little storage reserve in
the liver, and a deficiency can result when di-
etary intake of vitamin K is restricted or ab-
sorption is impaired.
Dietary vitamin K and supplements are
processed similarly as with the other fat-solu-
ble vitamins. Bile salts are required for emul-
sification and formation of mixed micelles.
They travel to the liver on chylomicrons along
with vitamins A and E.
Vitamins
0
II
C-NH-
I
-HN-CH
I
- 0
CH2
I
'32
Vitamin K Base I
co2-
\f GIU
I
I
/CH\
Vitamin K oxide -02C co2-
3.4.3 Vitamin K Biochemistry and Defi-
ciency. Deficiencies of this vitamin lead to se-
rious hemorrhaging. The vitamin is required
for formation of proteins that complex cal-
cium. This is done by functioning as a coen-
zyme in the y-carboxylation of glutamic acid
(Fig. 8.24). Vitamin K, is reduced to the hy-
droquinone. After several steps, a complex ox-
idation occurs, resulting in the "vitamin K
base" that is an integral part of the carboxyla-
tion step. In a key step, the vitamin K oxide is
reduced to the original vitamin K,. It is this
final reduction that is inhibited by the couma-
din anticoagulants widely used by patients
susceptible to stroke, pulmonary embolism,
phlebitis, and coronary thrombosis. This in-
teraction between the coumadin anticoagu-
lants and the regeneration of vitamin K, is the
reason that patients on coumadin must moni-
tor their vitamin K intake both from vitamin
supplements and diet. This usually is done by
regularly scheduled determinations of pro-
thrombin time.
The carboxylation reaction is required for
production of several clotting proteins includ-
ingprothrombin, protein C, protein S, and fac-
tors VII, M,and X. It is also required for y-
arboxylation of osteocalcin, an important cal-
d in the matrix of
ne and required for proper deposition of cal-
um onto bone. This latter finding has led to
everal studies to determine whether patients
d coumadin anticoagulants are at in-
risk for osteoporosis and fractures
All of these studies indicate that vi-
supplementation might be beneficial
evention of osteoporosis. If vitamin
revent osteoporosis, then it would
patients on anticoagulant therapy
Id be at increased risk for bone fractures.
e indication of this, but it is not
evertheless, some calcium sup-
both vitamins D and K added to
3.4.4 Causes of Vitamin K Deficiency.
ly is a vitamin K deficiency caused by in-
cient diet. It more likely is attributable to
n. At one time, multivita-
min supplements rarely contained vitamin K.
It is now routinely found in these products.
K deficiency include ob-
ctive jaundice (now uncommon), loss of in-
paration for intestinal sur-
gic disease of the newborn.
ed by obstructive jaundice
kage of the bile duct, usu-
ally from cholelithiasis, preventing the release
of bile salts into the intestinal tract for emul-
e prescribed because it
s not require micelle formation, given that
through the mucosa into
he liver. Alterna-
injections of phytonadi-
rgery of the intestinal
undergo 1-2 weeks'
ce the level of intes-
ient did not eat
ake of dietary vita-
K and vitamin from the intestinal bacte-
could result in a vitamin K deficiency. De-
termination of prothrombin time is ordered by
the surgeon to determine whether menadione
or phytonadione is indicated.
The third cause is hemorrhagic disease of
the newborn. Infants are born with a sterile
intestinal tract. Until the flora are estab-
lished, the infant will have to get along with
the vitamin K they received from the mother.
In the past an infant might die from hemor-
rhaging. Most states require that each new-
born receive an injection of phytonadione.
Menadione injection is not given because it
can cause a hemolytic anemia in the newborn.
3.4.5 Hypervitaminosis K. Although it is
possible to overdose with this vitamin, the fact
that it is available only over the counter in
small doses in multivitamin preparations has
resulted in little knowledge of any toxicities.
Toxicities do not appear in animals adminis-
tered large doses. It is known that excess in-
take of the vitamin does not promote clot for-
mation. There is no Tolerable Upper Intake
Level.
3.4.6 Dietary Reference Intakes.
AT
Infants 2-2.5 &day
Children (1-8 years) 30-55 pglday
Boys and Girls (9-18 years) 60-75 pglday ,
Men 120 pglday
Women 90 d d a ~
Pregnancy 75-90 pg/day
Lactation 75-90 pgtday
3.5 Thiamine (Vitamin B,) (43)
After 26 years of constant research, the vitamin
preventative of the disease beri-beri has been iso-
lated, its chemical constitution determined and
the vitamin itself synthesized at a cost far lower
than that of recovering it from bran.(Scientific
American, February 1938; reprinted in 258,12
February 1988)
This quote summarizes the debate between
those who believed that beriberi was caused by
an infectious agent vs. those who promoted
proper diet. Some of the early researchers os-
cillated between the two theories. About 1912
there were enough definitive feeding experi-
ments in humans to conclude that beriberi's
origin was dietary (44).

Figure 8.25. Commercial
forms of thiamine and for- H~C
mation of thiamine pyro-
phosphate.
The vitamin B complex, of which thiamine
is considered the first one discovered and char-
acterized, generally includes the group of wa-
ter-soluble vitamins found in rice polishings,
bean extracts, yeast, and liver. There are no
chemical relationships in the B complex. The
nomenclature is very confusing. The common
name originally implied something about the
chemical nature of the vitamin. Even the con-
cept of water-soluble is somewhat misleading,
given that some of the vitamins in this group
would be considered poorly soluble by most
pharmaceutical standards. The one thing the
B complex vitamins have in common is that
nearly all function either as a coenzyme or a
structural component of a coenzyme.
3.5.1 Chemistry. Thiamine consists of a
pyrimidine joined to a thiazole ring by a meth-
ylene bridge (Fig. 8.25). The thiazole nitrogen
is a quaternary with a permanent positive
charge. There are two commercial salts. Thia-
mine hydrochloride is, in reality, thiamine
chloride hydrochloride. It is a double salt, con-
Vitamins
Thiamine Mononitrate
Thiamine HCI
AMP
4
(Thiamine Chloride HCI)
Thiamine
kinase
Thiamine Pyrophosphate
sisting of an amine hydrochloride on the py-
rimidine amine and a chloride on the thiazole
quaternary nitrogen. Thiamine nitrate is cor-
rectly named in that the nitrate anion is found
on the quaternary nitrogen, and the pyrimi-
dine amine is not protonated. Once the vita-
min enters the acidic stomach, it will exist as
the chloride hydrochloride salt.
Thiamine hydrochlorideis very water soluble
(1g/l mL).It is also very hygroscopic, making it
difficult to use in dry formulations. This salt is
commonly used in liquid and injectable forrnula-
tions. Thiamine nitrate is sufficiently water sol-
uble (1 gl35 mL) that it can be used in liquid
formulations because the RDAs are less than 2
mg per day. Because it is nonhygroscopic, it is
commonly used in dry dosage forms.
3.5.2 Uptake and Metabolism. A saturable
active transport system in the jejunum pro-
vides efficient uptake of the vitamin into the
intestinal mucosa cell. Thiamine kinase in the
intestinal mucosa cell transfers a pyrophos-
phate from the ATP to the propyl alcohol at po-
sition 5 of the thiazole ring, forming thiamine cal literature and were called beriberi, a Japa-
pyrophosphate (TPP) (Fig. 8.25). The latter nese term. Sailors in the Japanese navy
product is the coenzyme form of the vitamin. experienced thiamine deficiencies when fed
There is some evidence that this phosphoryla- rice in which the polishings had been removed
tion is the rate-limiting step and controls the to prevent mold growth. This is somewhat
absorption of the vitamin. The coenzyme is analogous to removing the germ from wheat
transported to the tissues where needed. to prolong the shelf life of flour-containing
Thiamine pyrophosphate has two impor- foods. There are two forms of beriberi, wet and
tant coenzyme roles, both of which focus dry. Wet beriberi is characterized by edema
mostly on carbohydrate metabolism (Figs. and enlarged heart. Dry beriberi is more neu-
8.26 and 8.27). The active portion of the coen- rological and can include muscle wasting.
zyme is the thiazole ring. The first step in the
Assuming a reasonably balanced diet, most
oxidative decarboxylation of a-keto acids re-
thiamine deficiencies today are caused by
quires TPP. The two most common examples
are pyruvate and a-ketoglutarate, oxidatively chronic alcoholism. Alcohol reduces the active
decarboxyated to acetyl CoA and succinyl CoA, transport of the vitamin (45). This form of
respectively. The same reaction is found in the thiamine deficiency is called Wernicke-Korsa-
metabolism of the branched-chain amino ac- koff syndrome. It is common for emergency
ids valine, isoleucine, leucine, and methionine. medical personnel to add thiamine to the in-
In all cases, TPP is a coenzyme in a mitochon- travenous solution being administered to a co-
drial multienzyme complex, consisting of matose patient suspected of experiencing sub-
TPP, lipoic acid, coenzyme A, FAD, and NAD. stance abuse.
Note the number of vitamins required for the 3.5.4 Hypervitaminosis Thiamine. The vi-
oxidative decarboxylation of a-keto acids: thi- tamin is considered very safe. There are no
amine (TPP), pantothenic acid (coenzyme A),
Tolerable Upper Intake Levels. Possibly the
riboflavin (FAD),and niacin (NAD).
rate-limiting phosphorylation step in the in-
TPP is also the coenzyme in the transketo-
lase reaction (Fig. 8.27) found in the pentose testinal mucosa reduces the risk of toxicity.
phosphate pathway that interconverts hex- The percentage of thiamine absorbed de-
oses, pentoses, tetroses, and trioses. This reac- creases as the dose increases.
tion removes carbons 1 and 2 of a ketose and
transfers them to an acceptor aldose. Exam- 3.5.5 Dietary Reference Intakes.
ples include TPP transferring carbons 1 and 2 A1
of xylulosed-P to ribose-5-P, producing glyc- Infants 0.2-0.3 mgtday
eraldehyde-3-P (5 carbons minus 2 carbons) EAR
and sedoheptulose-7-P (5 carbons plus 2 car- Children (1-13 years) 0.60.7 mgtday
bons). This reaction is reversible. A second re- Males (14-18 years) 1.0 mglday
versible reaction has TPP transferring car- Females (14-18 years) 0.9 mgtday
bons 1 and 2 of xylulose-5-P to erythrose-4-P, Men (19-50+ years) 1.0 mglday
producing fructose-6-P (4 carbons plus 2 car- Women (19-50 + years) 0.9 mglday
bons) and glyceraldehyde-3-P (5 carbons mi- Pregnancy 1.2 mglday
Lactation 1.2 mglday
The dietary reference intakes for thiamine RDA
are dependent on carbohydrate consumption. Children (1-13 years) 0.5-0.9 mgtday
This is because (1)most pyruvate comes from Males (14-18 years) 1.2 mglday
aerobic glycolysis, (2) much of the decarboxy- Females (14-18 years) 1.0 mgtday
lated a-ketoglutarate originates from carbo- Men (19-50+ years) 1.2 mgtday
hydrate sources, and (3) the transketolase re- Women (19-50+ years) 1.1 mgtday
action uses carbohydrates as substrates. Pregnancy 1.4 mgtday
Lactation 1.5 mglday
3.5.3 Thiamine Deficiencies. Thiamine de- UL
ficiencies are reported in the very early medi- None reported
 -
/C-s TPP carbanion I
0

H
TPP CH20H HO- C> CH20H
t I
I
c=o H- 0-c- H
IU
HO-C-H
UH-C-OH
I
I
H- C-OH H+ I
CH20P
I +
CH20P
Xylulosed-P

HO-C-H
I
I
I H-c- OH
H-C-OH

I I
H-C-OH
H-C-OH
I
H-C-OH
I H-C-OH
I
I
CH20P
CH20P
Ribose-5-P
Sedoheptulose-7-P

Figure 8.27. Coenzyme role o f thiamine pyrophosphate in t h e transketolase reaction.

 Vitamins

3.6 Riboflavin (Vitamin B,) (46) conversion of folic acid and pyridoxine to their
active forms, it is surprising that riboflavin
Shortly after the discovery of thiamine from deficiency does not produce a characteristic
yeast concentrates, the presence of a second set of symptoms. One of the reasons may be
nutritional factor in such materials was sug- that it is rare to see a patient who is solely
gested. This second factor was also reported to deficient in riboflavin.
have a pellagra-preventative activity because
it alleviated a deficiency-induced dermatitis in 3.6.5 HypervitaminosisRiboflavin. The com-
rats. It was called vitamin B, in England and bination of regulated active transport and con-
vitamin G in the United States. version to the coenzyme forms prevents hy-
pervitaminosis problems with this vitamin.
3.6.1 Chemistry (Fig. 8.28). Riboflavin has Toxicities from the water-soluble riboflavin
a characteristic flavin ring system, which gives phosphate have not been reported. There are
it unique spectroscopic and instability proper- no Tolerable Upper Intake Levels.
ties. There are two commercial forms. Ribofla-
vin itself is poorly water soluble (1 gl10,OOO 3.6.6 Dietary Reference Intakes.
mL) and is limited to oral dry dosage forms.
Riboflavin phosphate, as the sodium salt, is AI
very water soluble at 100 mg/mL and is widely Infants 0.3-0.4 mglday
used in dry and liquid dosage forms. EAR
Children (1-13 years) 0.4-0.8 mglday
Males (14-19 years) 1.1 mglday
3.6.2 Riboflavin Uptake and Chemistry (Fig. Females (14-19 years) 0.9 mglday
8.28). Most dietary riboflavin is eaten as the Men (19-70+ years) 1.1 mglday
FAD or FMN coenzymes. Intestinal pyrophos- Women (19-70+ years) 0.9 mglday
phatases and phosphatases produce free ribo- Pregnancy 1.2 mglday
flavin, which is actively transported in the Lactation 1.3 mglday
proximal area of the small intestine into sys- RDA
temic circulation. Because of its poor water Children (1-13 years) 0.5-0.9 mglday
solubility, it is transported on albumin and Males (14-19 years) 1.3 mglday
immunoglobulin proteins. Conversion to the Females (14-19 years) 1.0 mglday .
coenzyme forms occurs inside the cells that Men (19-70+ years) 1.3 mglday
need these coenzymes. Women (19-70+ years) 1.1 mglday
Pregnancy 1.4 mglday
3.6.3 Metabolic Role. Riboflavin coen- Lactation 1.6 mglday
zymes are required for most oxidations of car- UL
bon-carbon bonds (Fig. 8.29). Examples in- None reported
clude the oxidation of succinyl CoA to
fumarate in the Krebs cycle and introduction 3.7 Niacin (Nicotinic AcidVNiacinamide
of a$-unsaturation in P-oxidation of fatty ac- (Nicotinamide) (47)
ids. Riboflavin is also required for the metab-
olism of other vitamins, including the reduc- The history of niacin revolved around trying
tion of 5,lO-methylene tetrahydrofolate to to find a way to prevent and cure pellegra, the
5-methyl tetrahydrofolate (Fig. 8.491, and in- late-stage deficiency disease caused by a niacin
terconversion of pyridoxine-pyridoxal phos- deficiency. Pellagra has been a serious nutri-
phate-pyridoxamine (Fig. 8.33). Because oxi- tional disorder in the United States, mostly in
dation/reductions that use FAD or FMN as the the southeast. Two thousand deaths from pel-
coenzyme constitute a two-step process, some lagra were reported in 1941. This is ironic be-
flavin coenzyme systems contain more than cause nicotinic acid, later known as niacin,
one FAD or FMN. was first reported during the structure eluci-
dation of the alkaloid nicotine.
3.6.4 Riboflavin Deficiency. With ribofla- Like some of the other deficiency diseases,
vin's central role in energy metabolism and there was disagreement between those who

0 plex reaction, quinolinic acid loses the 2-car-
FAD or FMN
(oxidized)
FADH- or FMNW
(semiquinone)
I 0
H
FADH2or FMNH2
(reduced)
Figure 8.29. FAD/FADH,-FMNPMNH, oxida-
tionlreduction.
thought pellegra was caused by poor sanita-
tion vs. those who concluded it was a nutri-
tional disorder. Niacin deficiency, even today,
is found in economically poor areas. Even
when niacin was first isolated from foods, it
was ignored because it did not cure beriberi.
3.7.1 Niacin/Niacinamide Chemistry, Up-
take and Metabolism. Niacin is structurally
the simplest of the vitamins, but has some of
the most complex biochemistry of the vita-
Vitamins
mins. Structurally, it is pyridine-3-carboxylic
acid (Fig. 8.30). Strictly speaking, it is nones-
sential because the essential amino acid tryp-
tophan is a source (Fig. 8.31) (48). The biosyn-
thetic route does not produce "free" niacin by
decarboxylation of quinolinic acid. In a com-
boxyl group and adds 5'-phosphoribose to
form nicotinate mononucleotide (Figs. 8.30
and 8.31) (49,50). Niacin and niacinamide, in
pharmaceutical dosage forms, undergo a sim-
ilar ribosylation reaction. Most vitamin prod-
ucts contain niacinamide because niacin can
cause a distracting vasodilation that leads to
flushing in the face and scalp.
The two commercial forms of the vitamin,
niacin and niacinamide, are rapidly absorbed
from both the stomach and intestine. As the
dose increases, absorption decreases. It is not
clear whether there is a feedback mechanism
operating or the transport system becomes
saturated. Conversion to the coenzyme forms
occurs in the cells where NAD and NADP are
needed.
NAD is the primary coenzyme required for
oxidation/reduction of carbon-oxygen bonds
and is required for oxidative catabolism (gly-
colysis, @oxidation,Krebs cycle). NADP is the
coenzyme in biosynthetic routes (fatty acid
and cholesterol synthesis) and will be part of
oxidation/reduction reactions involving both
carbon-oxygen and carbon-carbon bonds.
The active part of the coenzyme is the pyri-
dine ring (Fig. 8.32). When the substrate is
labeled with deuterium, it has been shown
that NAD systems can be categorized by the
deuterium ending up on the A or B face of the
pyridine ring. Examples of NAD dehydroge-
nases where the hydride anion attaches to the
A face are isocitrate dehydrogenase, malate
dehydrogenase, lactate dehydrogenase, and
alcohol dehydrogenase. Dehydrogenases in-
volving the B face include a-ketoglutarate
dehydrogenase, glucose-6-phosphate dehydro-
genase, glutamate dehydrogenase, and glycer-
aldehyde-&phosphate dehydrogenase.
3.7.2 Niacin Deficiency. Niacin deficiency,
manifested as pellagra, is characterized by the
four Ds: dermatitis, diarrhea, depression, and
death. The dermatitis is characterized by a
pigmented rash developing on skin exposed to
3 Vitamins

Trvptoph~

NkotWc Add (Niacin)

pwp
PPI
-jg;

ZATP + Gh + H P 1. NAD* pyrophorphoryla~

ADP + PPI + Glu

R =H
Nlmtinamlde adenine dlnudsot#a (IUD+);

Figure 8.30. Biosynthesis of NAD from niacin and niacinamide.

heat. Changes to the gastrointestinal tract can
lead to vomiting, constipation, or diarrhea.
Depression is one of the neurological symp-
toms that also can include apathy, headache,
fatigue, and memory loss.
Deficiencies were common in populations
whose main calorie source was corn (51, 52).
Zein is the main protein found in corn and it is
very low in both niacin and tryptophan. When
corn is ground with lime water, the small
amounts of niacin and tryptophan become more
bioavailable. Populations who consume corn
meal by this process have a smaller incidence of
pellagra (53). At the same time it must be re-
membered that pellegra is the extreme form of a
niacin deficiency. The Dietary Reference In-
takes for this vitamin use clinical chemistry as-
says listed in Table 8.2 to determine DRIs rather
than the first appearance of pellagra symptoms.
3.7.3 Hypervitaminosis Niacin. Niacin is
considered nontoxic, and there are no Tolera-
ble Upper Intake Levels based on its use as a
vitamin. These refer only to niacin and niaci-
 Vitamins

CH2-CH-
I I
C02-
I
NH3+ *
TryptophanP,3-
dioxygenase
H
Tryptophan Formylkynurenine

Kynurenine
formamidase

- Kynurenine
CH-
I
NH$
C02-

hydroxylase
(FAD dependent)
Kynurenine
Kynureninase

Co2- 3-Hydroxyanthranilic C02- non-

acid oxidase enzymatic

OH Amino-carboxymuconic Quinollinicacid
3-Hydroxyanthranilic Acid semialdehyde
i
NAD

Figure 8.31. Biosynthesis o f NAD f r o m tryptophan.

namide, but not tryptophan and niacin equiv-
dents (see next section). Large doses of niacin
and niacinamide do have adverse reactions.
These are sometimes seen with patients who
are prescribed niacin in doses up to 2 g daily
for hyperlipidemia, including both hypercho-
lesterolemia and hypertriglyceridemia. For
the former, there is the desired decreased LDL
and increased HDL. Adverse reactions include
vasodilation from niacin, particularly in the
head area, caused by increased intercranial
blood flow and hepatic complications. Niacin
had been used for Raynaud's syndrome to
treat the vasoconstriction seen with this dis-
ease. Liver toxicities have been experienced by
patients prescribed sustained-release niacin
products for hyperlipidemia (54, 55). The UL
to RDA ratio is 2, but the UL is the dose before
adverse reactions are experienced. Vasodila-
tion from niacin occurs close to its RDA.

0 0 0

N H2 N H2
or
N+
I DF
I I
R R R
NAD+ A face; NADH B face; NADH

Figure 8.32. Stereochemistry o f NAD reduction.

3 Vitamins

3.7.4 Dietary Reference Intakes. Many of was realized tragically when the heat process
the DRI units are milligram niacin equiva- for an infant formula reduced the bioavailabil-
lents (mg NE). These units take into account itiy of the vitamin. Children developed convul-
the fact that approximately 60 mg trypto- sive disorders. Before realizing that the prob-
phan produce 1 mg of niacin. For adult lem was caused by an induced pyridoxine
males, the RDA is between 960 mg of tryp- deficiency, it was thought a contaminant had
tophan and 16 mg of niacin, given that 960 been introduced. The vitamin is a coenzyme
mg of tryptophan is equivalent to 16 mg of for amino acid and glycogen metabolism.
niacin (56, 57).
A1 3.8.1 Uptake and Metabolism. The vitamin
Infants (0-5 months) 2 mglday of B6 family consists of pyridoxine, pyridoxal,
preformed pyridoxamine, pyridoxine phosphate, pyri-
niacin doxal phosphate (PLP), and pyridoxamine
Infants (6-11 months) 4 mg NE/day phosphate (Fig. 8.33). The commercial form is
EAR pyridoxine. Pyridoxal phosphate is the coen-
Children (1-13 years) 5-9 mg NEIday zyme form. It and pyridoxamine phosphate
Boys (14-18 years) 12 mg NEIday are from animal tissues. Pyridoxine is from
Girls (14-18 years) 11 mg NEIday plant tissues. All phosphorylated forms are
Men (19-70+ years) 12 mg NEIday hydrolyzed in the intestinal tract by phospha-
Women (19-70+ years) 11 mg NE/day tases before being absorbed passively. Conver-
Pregnancy 14 mg NEIday sion to the phosphorylated forms occurs in the
Lactation 13 mg NEIday liver. Notice that niacin (NAD) and riboflavin
RDA (FMN, FAD) are required for interconversion
Children (1-13 years) 6-12 mg NEIday among the vitamin B6 family. The phosphory-
Girls (14-19 years) 1.3 mg NEIday lated forms are transported to the cells where
Boys (14-19 years) 16 mg NEIday needed. The major excretory product is 4-pyr-
Men (19-70+ years) 16 mg NEIday idoxic acid.
Women (19-70+ years) 14 mg NE/day
Pregnancy 18 mg NEIday 3.8.2 Pyridoxal Phosphate Biochemistry
Lactation 17 mg NEIday (58). Pyridoxal phosphate (PLP) is required
UL for amino acid metabolism and reactions in-
Infants (0-12 months) Source of intake volving amino acids. PLP is covalently bound
should be to the apoenzyme through an enamine
formula and (Schiff's base) linkage between an €-amino
food only group of lysine and the aldehyde moiety of
Children (1-13 years) 10-20 mglday of PLP (Fig. 8.34). The most common of the
niacin PLP-catalyzed reactions are transaminations
Adolescents (14-18 30 mglday of (Fig. 8.35). One-half of all transamination re-
years) niacin actions involve a-ketoglutarate as the accep-
Pregnancy (14-18 30 mglday of tor of the m i n e group forming glutamic acid.
years) niacin Alternatively, glutamic acid donates the
Pregnancy (19 years 35 mglday of amine group and an a-keto acid is the acceptor
and older) niacin forming a new amino acid. Examples include:
Lactation (14-18 30 mglday of a-amino acid + a-ketoglutarate % a-keto
years) niacin acid + glutamic acid
Lactation (19 years and 35 mglday of a-amino acid reactant a-ketoacid product
older) niacin alanine pyruvate
aspartate oxalacetate
3.8 Vitamin B, Family
Another PLP-catalyzed reaction is decarbox-
This group was discovered in the 1930s during ylation of amino acids (Fig. 8.35). These are part
feeding experiments on rats. Its importance of the biosynthesis of neurotransmitters, includ-
 Vitamins

coo-

4-Pyridoxic acid
(metabolite)

Oxidase

CH20H CHO CH2NH3+

I

H+ CH20H
Oxidase - CH20H

Oxidase -7-i-
FMNHz FMN H~C
H3C N
4-Pyridoxic acid 4-Pyridoxic acid 4-Pyridoxic acid
(metabolite) (metabolite) (metabolite)

Kjpi
Phosphatase %Le
ATp Phosphatase
Kj
pi #Kse
ATp
pi
Phosphatase jk ELe
ATp

CH20H CHO CH2NH3+

CH20P CH20P
FMN FMNH2
Oxidase
Oxidase
H3C N FMNH2 FMN H3C
4-Pyridoxic acid 4-Pyridoxic acid 4-Pyridoxic acid
(metabolite) (metabolite) (metabolite)

Figure 8.33. Interconversions among the vitamin B, family.

.

ing histamine from histidine; serotonin from

tryptophan, dopamine, norepinephrine, and epi-
nephrine from dihydroxyphenylalanine (dopa)
0

!
(Fig. 8.36); and y-aminobutyric acid from glu-
II tamic acid. Other reactions in which an amino
C JVV
I acid is the substrate include formation of Gami-
Enzyme mN-C-H nolevulinic acid in the biosynthesis of heme and
1 two reactions in cysteine biosynthesis from
(CH2)4 methionine (Fig. 8.52). Although not well un-
I derstood, PLP is required for phosphorylase-
catalyzed
phosphate.glycogenolysis producing glucose-l-
?&
CH20P
3.8.3 Vitamin B, Deficiency. There is no
H3C N distinct deficiency syndrome, but a deficiency
Pyridoxal P can be serious. What is observed is a sebor-
rheic dermatitis, microcytic anemia, and elep-
Figure 8.34. Wdoxal phosphate bound to the tiform convulsions. The anemia may be
enzyme. caused by decreased heme biosynthesis and
I1
b
3 Vitamins
:

1
i I H R-C-C02-
i R-C-C02-
j
I I II
1
I NH3+ R-C-C02- R-C-C02- 0
a-amino acid Hz0
N
I II a-keto acid
!
+
r
j H 4 I1
i C

Pyridoxal P Aldimine
Pyridoxamine P

I
R-C-H
I
NH+
II
CH
CH20P

NH3+
Amine
Figure 8.35. Pyridoxine phosphate-catalyzed transamination and decarboxylation.

the convulsions by imbalances in neurotrans-
mitter biosynthesis.
There is reason to conclude that vitamin B,
deficiency might contribute to arteriosclerosis.
There is a correlation between elevated homo-
cysteine levels and incidence of cardiovascular
disease (59). There is debate as to whether ho-
mocysteine contributes to the damage of cells on
the interior of blood vessel or whether homocys-
teine is a marker of intensive cell repair and for-
mation of replacement cells. Nevertheless, ad-
ministration of pyridoxine, folic acid, and
cyanocobalamin are being recommended along
with the two antioxidant vitamins, a-tocopherol
and ascorbic acid for arteriosclerosis. Vitamin
B, is required for two of the steps in the catabo-
lism of homocysteine to succinyl CoA (Fig.8.52).
Note in Fig. 8.52 (bottom) that biotin and a co-
enzyme f o m of cobalamin also are required for
the final reactions. The homocysteine model is
discussed again under the folic acid and cyano-
cobalamin sections.
3.8.4 Vitamin-Drug Interactions (60). The
two most clinically significant interactions are
pyridoxal phosphate with L-dopa or isoniacid.
Examine Fig. 8.36 and note that dopa decar-
boxylase requires PLP. This enzyme is found
both centrally and peripherally. The latter in-
cludes the intestinal mucosa. The precursor to
dopamine, L-dopa is indicated for the treat-
ment of Parkinson's disease. L-Dopa is pre-
scribed because little dopamine crosses the
blood-brain barrier relative to its precursor L-
dopa. A patient with Parkinson's disease pre-
scribed L-dopa and who takes a vitamin sup-
plement with amounts of pyridoxine greater
than the vitamin's RDA can experience an in-
 Vitamins

peripheral neuropathy, but they do not reduce

the effectiveness of isoniazid.

3.8.5 Hypervitaminosis Pyridoxine. A cer-

tain mystique has built up around this vita-
min, resulting in individuals' overdosing
themselves with commercial vitamin supple-
ments. Serious neurological problems have
been seen in doses of 2-6 glday for 2-40
months (62-64). Megadosing below 2 glday
seems safe, but all of this information is based
mostly on anecdotal reports. There is a Toler-
able Upper Intake Level, but the UL to RDA
ratio is a comfortable 50-60.

HO
3.8.6 Dietary Reference Intakes.
Dihydroxyphenethylarnine (Dopamine)
A1 (any form of vitamin B,)
Ascorbate + O2 Dopamine Infants 0.1-0.3 mglday
phydroxylase
EAR (any form of vitamin B,)
Dehydroascorbate + H20 l/i Children (1-13 years) 0.4-0.8 mglday
Males (14-19 years) 1.1 mglday
Females (14-19 years) 1.0 mglday
Men (19-50 years) 1.1 mglday
Men (51+ years) 1.4 mg/day
Women (19-50 years) 1.1 mglday
Norepinephrine (Norepi) Women (51+ years) 1.3 mglday
Pregnancy 1.6 mglday
Lactation 1.7 mglday
RDA (any form of vitamin B,)
t Children (1-13 years) 0.5-1.0 mglday
NH3' Males (14-19 years) 1.3 mglday
I Females (14-19 years) 1.2 mglday

H0PyH-cH
HO OH

Epinephrine (Epi)
Men (19-50 years)
Men (51+ years)
Women (19-50 years)
Women (51+ years)
1.3 mglday
1.7 mglday
1.3 mglday
1.5 mglday
Pregnancy 1.9 mglday
Figure 8.36. Pyridoxal phosphate-catalyzed dopa Lactation 2.0 mglday
decarboxylase. UL (as pyridoxine)
Children (1-13 years) 30-60 mglday
crease in parkinsonian tremors. This is be- Adolescents (14-18 years) 80 mglday
cause L-dopa will undergo decarboxylation in Adults (19+ years) 100 mglday
the intestinal mucosa and never reach the lo- Pregnancy (14-18 years) 80 mglday
cations in the brain where it is converted to Pregnancy (19+ years) 100 mglday
the needed dopamine. Lactation (14-18 years) 80 mg/day
Isoniazid is widely prescribed for tubercu- Lactation (19+ years) 100 mglday
losis. It can chemically react with pyridoxal
and pyridoxal phosphate, thus significantly re- 3.9 Pantothenic Acid (65)
ducing the availability of this coenzyme (Fig.
8.37) (61). Pyridoxine supplements commonly Pantothenic acid is essential and is a normal
are recommended to prevent isoniazid-caused component of our diet. There has been little
3 Vitamins
lsoniazid (INH) Pyridoxal P
lsonicotinic Acid Hydrazide
al Adduct
lsoniazid Pyridoxal
research done on this vitamin and, therefore,
it has adequate intakes (AI) and no RDAs.
3.9.1 Chemistry, Uptake, and Metabolic
Role. This vitamin, which can be considered a
derivative of p-alanine, is asymmetric (Fig.
8.38). The natural form has the D(+)configu-
ration. The L(-)stereoisomer is inactive. The
reduced alcohol form, pantothenol, is consid-
ered as equally active as the parent acid. Many
of the multiple vitamin products use a syn-
thetic, racemic mixture. This means that dou-
ble the amount of synthetic vitamin must be
used to obtain equivalent active vitamin.
Dietary pantothenic acid is consumed as co-
enzyme A and the intermediates from coen-
zyme A's biosynthesis (Fig. 8.39). These are
hydrolyzed to free pantothenic acid. Absorp-
tion is by saturable, active transport.
Calcium pantothenate is commonly used in
dry dosage forms. It is moderately hygro-
scopic, with a solubility of 1 gl2.8 mL, and is
unstable for autoclaving. Neither the parent
pantothenic acid nor the sodium salt is com-
monly used in dosage forms.
Pantothenol (panthenol)is reasonably stable
and freely soluble and is used both in injectable
and oral dosage forms. Although widely used in
Figure 8.37. Pyridoxal phosphate-isoniazid
interaction.
cosmetics including skin creams and shampoos,
there is no evidence that this vitamin is effective
as a vitamin topically. It apparently has good
emollient properties, but these have nothing to
do with its systemic role.
Pantothenic acid is a structural compo- .
nent, but not the active site, of coenzyme A.
The acyl thiol esters form on the mercaptan
moiety that originates from a cysteine (Fig.
8.39). The biosynthesis of coenzyme A occurs
in the tissues requiring it. Because coenzyme
A is required for nearly all acyl transfers, bio-
synthesis takes place in nearly all cells.
3.9.2 Hypervitaminosis Pantothenic Acid.
There have been no reports of toxicity and no
Tolerable Upper Intake Levels. Because its ac-
tive transport is saturable, excessive uptake is
doubtful. Also, this vitamin does not have the
mystique that would prompt marketing "high
potency" formulations.
3.9.3 Dietary Reference Intakes. There are
too few studies to provide sufficient informa-
tion to estimate Estimated Average Require-
ments (EAR) or Recommended Dietary Allow-
ances (RDA).
 Vitamins

0 0 OH CH3
II II I I
HO-C-CH2-CH2-NH-C-CH-C-CH20H
I
CH3
Pantothenic acid

CH3
Sodium Pantothenate

1 CH3 1 2

Calcium Pantothenate

0 OH CH3
I1 I I
HO-CH2-CH2-CH2-NH- C-CH- C-CH20H
I
Figure 8.38. Forms of panto-
thenic acid. Pantothenol

amide linkage to produce free biotin. Biotin is

A1
Infants 1.7-1.8 mglday actively transported through the intestin@
Children (1-13 years) 2 4 mglday flora into the portal vein and to the liver where
Everyone else 5 mglday it may be stored. It appears that adults may
Pregnancy 6 mglday store several months of biotin. From the liver
Lactation 7 mglday it is transported to tissues where it is needed.
EAR 3.10.2 Chemistry. Biotin consists of two
None reported
5-membered rings cis-fused to each other that
RDA
can be drawn either as the keto (urea) or enolic
None reported
form (Fig. 8.40). The enolic d-isomer is the
UL
None reported active stereoisomer, but many times commer-
cial multivitamin products contain the syn-
3.10 Biotin (66) thetic racemic d,l mixture. There is no activity
with the 1-stereoisomer.
Biotin (Fig. 8.40) is essential, a normal constit-
uent of the diet, and required for four biotin- 3.10.3 Metabolic Role. Biotin picks up
dependent carboxylation reactions. Eating carbon dioxide that has been activated by
raw egg white can induce a deficiency. combining with an ATP-donated phosphate,
producing the mixed anhydride of phos-
3.1 0.1 Uptake. In foods, most biotin is co- phoric and carbonic acids (Fig. 8.42). The
valently bound (Fig. 8.41) to the apoenzyme, biotin enolate receives the carbon dioxide,
where it is the coenzyme for carboxylation re- producing the keto carbon dioxide-releasing
actions. Intestinal enzymes hydrolyze the coenzyme.
 H
-
OH
H -
OH
ATP ADP
-02c OH
LLL -O2C,CH2 \
OPO,-~
Pantothenate
0 kinase

Pantothenic acid 4'-Phosphopantothenate

ATP + Cys ,
ADP + Pi
Phosphopantothenoylcysteine
synthetase

Phosphopantothenoylcysteine
decarboxylase

2 ATP 4 1. Dephospho-CoA pyrophosphorylase

2. Dephospho-CoA kinase
ADP + PPi

H OH 0 0
H - II 11
\ CH2-0-P-0-P-0
I I
0 0- 0-

Coenzyme A (CoASH)

Figure 8.39. Pantothenic acid chemistry.

Enolic form Keto or ureido form Figure 8.40. Biotin.

404
Figure 8.41. Coenzyme form of biotin.
There are four biotin-dependent carboxyla-
tion reactions, three of which are in the mito-
chondria. They are:
1. Pyruvate carboxylase (Fig. 8.43)
This reaction, which converts pyruvate to
oxalaceetate, is in the mitochondria and
hastwo functions. First, it is the initial reac-
tion in gluconeogenesis to overcome the 14
kcal energy barrier to form phosphoenol-
pyruvate. Second, this same reaction,
sometimes referred to as an anapleurotic re-
action, ensures that there is adequate oxala-
Bicarbonate
0 0
II II
HO-P-0-C-0-H
I
0-
Phosphoric-carbonicacid anhydride
Figure 8.42. Biotin binding carbon dioxide.
Vitamins
cetate when there are large amounts of acetyl
CoA entering the Krebs cycle.
2. Acetyl CoA carboxylase (Fig. 8.44)
This reaction, found mostly in the cy-
tosol, is the committed step in the synthesis
of fatty acids.
3. Propionyl CoA carboxylase (Fig. 8.45)
Propionyl CoA is the product from the
catabolism of valine, isoleucine, methio-
nine, and odd-numbered fatty acids. The
carboxylation reaction, found in the mito-
chondria, produces methyl malonyl CoA.
The latter undergoes a cobalamin (vitamin
BIZ)-catalyzed rearrangement, forming
succinyl CoA, which is metabolized further
in the Krebs cycle.
4. pMethylcrotony1 &A carboxylase (Fig. 8.46)
This mitochondria1 reaction permits
the final steps in the catabolism of the
branched-chain amino acid leucine. The fi-
nal products, acetoacetate and acetyl CoA,
either are oxidative metabolized to carbon
dioxide and water or enter other reactions
in lipid metabolism.
0
Enz
 3 Vitamins

Pyruvate ATP ADP +Pi Oxalacetate Figure 8.43. Carboxylation of pyru-

Pyruvate carboxylase vate producing oxalacetate.

3.10.4 Biotin Deficiency. Relative to many 3.1 1 Folic Acid (67)

of the vitamins, it is easy to induce a biotin
deficiency by feeding volunteers raw egg
white. Avidin, a basic protein found in egg
white, forms salt linkages with acidic biotin
and prevent its transport across the intestinal
barrier. Cooked egg white is not a problem.
Because biotin is found in the yolk, eating
whole raw egg will not induce a deficiency. De-
ficiencies also were caused in patients on total
parenteral nutrition (TPN) because biotin
was not included in the early formulations.
Symptoms include dermatitis, loss of hair
color, and central neurological effects.
3.10.5 Hypervitaminosis Biotin. None has
been reported in humans and there are no Tol-
erable Upper Intake Levels.
3.10.6 Dietary Reference Intakes. These
have been difficult to determine. There has
been some speculation that humans might ob-
tain part of their biotin requirements from the
intestinal flora in the colon. The question that
has not been adequately answered is whether
there is significant absorption of bacteria-pro-
duced biotin from the colon.
3 A1
I1 ~nfants 5-6 pglday
Children (1-13 years) 8-20 pglday
Adolescents (14-18 years) 25 /&day
Adults 30
Pregnancy 30 p g l d a ~
Lactation 35
EAR
None reported
RDA
None reported
UL
None reported
Because all vitamins are essential, it is diffi-
cult to state that one vitamin is more impor-
tant than another. Nevertheless, folic acid,
with its coenzyme role in purine biosynthesis,
can be considered crucial for some of the cells'
most fundamental biochemistry, cell division.
This vitamin is intimately tied to vitamin B,,
(cobalamin), which has made estimating its
DRIs difficult. Also, conditions that can cause
a folic acid deficiency also can result in a vita-
min B,, deficiency.
3.1 1 .I Chemistry. The commercial form of
the vitamin is folic acid (Fig. 8.47). It consists
of a pteridine ring attached to a p-aminoben-
zoic acid that is attached to the a -m i n e of
glutamic acid. Two biosynthetic changes must
occur before it is active. First, it must be re-
duced to tetrahydrofolate by dihydrofolate re-
ductase in a two-step reduction (Fig. 8.48).
Notice that niacin is required for this reduc-
tion. Second, a polyglutamate chain must be
attached to the y-carboxyl of the parent glu-
.
tamic acid (Fig. 8.47). The remaining linkages
of the polyglutamate chain are traditional
a-amino-a-carboxyl peptides.
The natural vitamin is made up of a family
of polyglutamates all connected to the initial
glutamic acid at the previously described
y-carboy1 group. The length of this polyglu-
tamate chain varies with the source of the vi-
tamin, but lengths of 3, 5, and 7 amino acids
are seen. The most common polyglutamate
found in food is 5-methyltetrahydrofolate
polyglutamate (Fig. 8.47).
3.1 1.2 Uptake. The dietary polygluta-
mates are cleaved to the monoglutamate vita-

CH3-
0
II
C- SCOA
Acetyl CoA
bDATP
Biotin

ADP + Pi
0
11
0-C-CH2-C-SCoA
Malonyl CoA
11
Figure 8.44. Carboxylation of
acetyl CoA producing malonyl
Acetyl CoA Carboxylase CoA.
 CH3- I'
0

CH2- CSCOA
Propionyl CoA

Figure 8.45.
*- ATP
Biotin

ADP + Pi
Propionyl CoA carboxylase
0
11
0-C-CH-C-SCoA
CH3 0
I I1
Methyl Malonyl CoA

Carboxylation o f propionyl CoA producing methylmalonyl CoA.

Vitamins

min by a y-L-glutamylcarboxy peptidase, com- folate polyglutamate, produced by addition of

monly called conjugase. Folic acid is absorbed
as the monoglutamate. Conjugase is found in
the brush border of the intestine. Therefore,
chronic inflammatory conditions in the intes-
tine lead to low conjugase activity, which can
result in significant decreased folic acid
absorption.
The absorbed vitamin must be converted to
the coenzyme form. This requires adding back
a five- to seven-member glutamate chain and
commercial folic acid must undergo the two-
step reduction to tetrahydrofolic acid. There
are two common abbreviations for the reduced
form, FH, and THF. These reactions apparently
occur in a wide variety of tissues. The liver con-
tains about a 3- to &month supply of the vita-
min, presumably in the polyglutamate form.
3.11.3 Metabolic Roles. There are five
forms of tetrahydrofolate polyglutamate,
some of which are coenzymes (Fig. 8.49). The
most highly oxidized is 10-formyl tetrahydro-
formic acid to tetrahydroformate polygluta-
mate. It is the coenzyme for two reactions in
purine biosynthesis: ( 1 ) the synthesis of
formylglycine ribotide (FGAR) from glycine
amide ribotide (GAR) and (2)the formation of
aminoimidazole carboxamide ribotide (AICAR)
to formamidoimidazole carbxamide ribotide
(FAICAR) (Fig. 8.50).
5,lO-Methenyl tetrahydroformate polyglu-
tamate is the cyclic enamime formed from 10-
formyl tetrahydroformate polyglutamate. It
also is formed from the catabolism of histidine
and can be considered an intermediate be-
tween the 10-formyl compound and, upon re-
duction, 5,lO-methylene tetrahydroformate
polyglutamate. The latter is the coenzyme re-
quired for the interconversion of serine and
glycine and the methylation of deoxyuridylic
acid forming deoxythymidylic acid (Figs. 8.49
and 8.51). Most of the one-carbon units car-
ried on position 5 or 10 or 5,10 bridge come
from serine.

COO- 0 -

I II cII -SCoA
+H3N-CH C -SCoA
I I
I Several steps I p-Methylcrotonyl I
CH2 *
.....-------
CH CoA carboxvlase CH
.~ 0
I .I1. II I1
C C /c\
H~C' CH3 ' ATP + COz ADP+ Pi H3C
/ \
CH2 0-
Leucine p-Methylcrotonyl CoA p-Methylglutaconyl CoA

Acetoacetate Acetyl CoA

Figure 8.46. Role o f b i o t i n in leucine catabolism.
II 3 Vitamins

p-aminobenzoate glutamate
Folic Acid (FA)

Figure 8.47. Common forms of folic acid and methotrexate.

The most reduced coenzyme is 5-methyl The fifth tetrahydrofolate compound is

tetrahydrofolate polyglutamate. It is the
source of the methyl group added to homocys-
teine regenerating methionine and tetrahy-
drofolate ready to accept a one-carbon unit
from formate or serine. This last reaction is
where folic acid and vitamin B,, come to-
gether (Figs. 8.49, 8.52, and 8.53). The impli-
cations of this reaction and how folic acid can
mask pernicious anemia are discussed in the
section on vitamin B,, (cyanocobalamin).
Note that the formation of 5-methyl-THFnor-
mally is not reversible. Tetrahydrofolate can
be regenerated only if there is adequate
methyl cobalamin coenzyme.
5-formyl THF (folinic acid, citrovorum factor).
This compound is not a coenzyme, but it can
be converted to any of the active coenzyme
forms. It is administered after treatment with
the dihydrofolate reductase inhibitor, metho-
trexate (Fig. 8.47), as a form of rescue therapy.
Because it already is in the reduced tetrahy-
drofolate form, it does not need dihydrofolate
reductase to become an active coenzyme.
3.1 1.4 Folic Acid Deficiency. It is obvious
that folic acid is a very important vitamin for
biosynthetic reactions, particularly those re-
quired for the biosynthesis of purines, methyl-
 Vitamins

0 COO-

Folic Acid (FA) I

COO-
NADPH + H+
Dihydrofolate
reductase
NADP+
H H
N,
coo-
I
CH2-NH C-NH-CH
OH I
(CH212
Dihydrofolate (DHF; FH2) 1
coo-
NADPH + H+
Dihydrofolate
reductase
NADP+

(CH2)2
Figure 8.48. Reduction o f folic Tetrahydrofolate (THF; FH4) I
coo-
acid to tetrahydrofolate.

ation of deoxyuridylic acid, and regeneration
of methionine from homocysteine. The main
deficiency is a characteristic megaloblastic
anemia attributed to a shortage of nucleotides
required for the production of erythrocyte pre-
cursor cells.
Another clinical sign of folic acid deficiency
is neural tube defects, including spina bifida
and anencephaly. Neural tube defects consti-
tute one of the main reasons that federal reg-
ulations mandate supplementing cereal grain-
based foods with folic acid along with
thiamine, riboflavin, and niacin (68, 69). Al-
though prenatal multiple vitamins contain ad-
equate amounts of folic acid, pregnant women
may not start taking these products until the
second or third month of the their pregnan-
cies, and this may be too late.
A third indication of inadequate folic acid is
elevated blood homocysteine levels, with atten-
dant increased risk of cardiovascular disease.
This hypothesis is based on an observation that
individuals with increased blood vessel plaque
buildup also show increased levels of homocys-
teine. The elevated homocysteine can be cor-
rected, at least partially, with folate supple-
ments. Figure 8.52 illustrates why the three
vitamins, pyridoxine, folic acid, and vitamin B,,,
are indicated for elevated levels of homocys-
teine. Pyridoxal phosphate (PLP) and cobal-
amin (B,,) are required for the catabolism of
homocysteine to succinyl CoA. Methyl cobal-
amine (methyl B,,) is required for the conver-
sion of homocysteine back to methionine.
There are many causes of folic acid defi-
ciencies. Inadequate nutrition during periods
of increased requirements is one of the main
causes of megaloblastic anemia of pregnancy
and neural tube defects. Alcoholism is consid-
ered the leading cause of folic acid deficiency
3 Vitamins

N5,N10-~ethylene-~~F

NADPH +H
H H

His
5 ,CH
'CH~
0 HN,
I10 0 HC-NIO
THF \
R
N5,N10-~ethenyl-THF

H
ADP + Pi
H

0 ~CHHNIO
5,CH 0 \
R
0 HC-Nio N5-Formyl-THF
// \ (Folinic acid'
0 R citrovorum factor)
N~~-FO~~~I-THF

Figure 8.49. Formation o f f o l k acid coenzymes.

in the United States (70, 71). There are two
ways that excessive alcohol consumption can
interfere with folic acid activity: impairment
of folic acid reduction to the active THF forms
and interference with folic acid storage and
release from the liver. A third cause of folic
acid deficiency is chronic inflammation of the
intestinal mucosa. Inflammation can reduce
production of the required conjugase enzyme,
which removes the polyglutamate chain,
 ~ ~

H2N\CH2 lN'cH2
I O=CH I
0 0
II II
-0-P-0-CH2 -0-P-0-CH2
GAR Transformylase
I I
0- 0-
OH OH I OH OH
~ ' ~ - ~ o r m y l - T H FDHF 5-Phosphoformylribosylglycinamide (FGAR)
5-Phosphoribosylglycinamide (GAR)
0
11
{ ~ " \ N H ~
NADPH
NADP+
+

4
H+ Dihydrofolate
reductase

THF
0
II
- 0- P- 0
NH2
-0 -P -0
0
II t& N '0

xql
AICAR Transformylase
I I
0-
OH OH
5'-Phosphoribosyl-5-aminoimidamle-4-
N'O-~ormylTHF DHF
O- KdOH OH
5'-Phosphoribosyl-5-formamidoimidazole-4-
carboxamide (AICAR) carboxamide (AICAR)

THF

Figure 8.50. Coenzyme roles for 1 0 - f o m y l THF.

 Vitamins 411

OH H t
N5.Ni0-Methvlene
.... ~ , THF DHF
Deoxyuridylic acid Deoxythymidylic acid
reductase
NADP+
THF

Figure 8.51. Methylation of deoxyuridylic acid forming deoxythymidylic acid.

and/or an inflamed mucosa inhibits folate 3.1 1.6 Dietary Reference Intakes.
transport. Finally, anticonvulsants such as A1
phenytoin somehow interfere with folic acid Infants
uptake or utilization. EAR
The dihydrofolate reductase inhibitor, Children (1-8 years) 120-160 &day
methotrexate (Fig. 8.47), was developed as an Children (9-13 years) 250 pglday
anticancer drug, whose inhibition of forma- Adolescents (14-18 years) 330 pg/day
tion of folic acid coenzymes would block pu- Adults (19-50+ years) 320 pglday
rine synthesis. In other words, it was designed Pregnancy 520 d d a y
to induce a folic acid deficiency. Notice in Figs. Lactation 450 pglday
8.50 and 8.51 that formation of dTMP, FGAR, RDA
and AICAR also causes the oxidation of tetra- Children (1-8 years) 150-200 pglday
hydrofolate to dihydrofolate. The latter must Children (9-13 years) 300 pglday
be reduced by dihydrofolate reductase to tet- Adolescents (14-18 years) 400 pglday
rahydrofolate before active coenzyme can Adults (15-50+ years) 400 &day
form again. Thus, not only does methotrexate Pregnancy 600 &day
inhibit the initial formation of the tetrahydro- Lactation 500 pglday
folate moiety, it blocks regeneration of the co- UL (from fortified food or
enzyme form. supplements)
Children (1-3 years) 300 pglday
3.1 1.5 Hypervitaminosis Folic Acid. This Children (4-8 years) 400 pglday
apparently is not a problem. Transport across Children (9-13 years) 600 pglday
the intestinal mucosa may be regulated by a Adolescents (14-18 years) 800 pglday
feedback mechanism or the rate of hydrolysis Adults (19+ years) 1000 pglday
of the polyglutamate chain or a combination of Pregnancy (14-18 years) 800 &day
both. What is very important is that taking the Pregnancy (19t years) 1000 pglday
vitamin in doses above 400 pg (800 pg in preg- Lactation (14-18 years) 800 &day
nant and lactating women) can mask the mac- Lactation (19+ years) 1000 pg/day
rocytic anemia seen with pernicious anemia
caused by a cyanocobalamin deficiency (Fig.
8.53). The Tolerable Upper Limit is based on 3.12 Vitamin B,, (Cobalamin) (72)
trying to avoid this masking. Therefore, the This chemically very complex vitamin is re-
UL to RDA ratio is low (2-2.5) in adults. quired for two reactions, the methylation of ho-
 L
ATP PPi + Pi

~ethionine-
adenosyl
transferase
I
CH2
I
CH2
I

OH OH
t'fJ
N
I N/

- wH
"methyl transferases"

R
iR-CH3
(carnitine. choline.
'rnethlated DNA, '
"
I
CH2
I
CH2
I

CH3
OH
tfJ
N

OH
I
N/

Methione S-Adenosylmethionine (SAM) epinephrine) S-Adenosylhomocysteine (SAH)

0
II
C- SCoA
I Several Cysthionine
CH2
steps ylyase
I +......------....
1. Biotin
CH2 -*
I 2. BI2 coenzyme NH4+ H20
C-O- -
11 SH NH3+ 0
0 I I II
CH2-CH-C-0-
Succinyl CoA Cystathionine
Cysteine

Figure 8.52. Methionine metabolism.

3 Vitamins
5,l 0-CH2-THF
::
5-CH3-THF
coenzyme
Homocysteine
THF
Met
-
DHF Reductase
Figure 8.53. Methyl trap hypothesis.
mocysteine to methionine and rearrangement of
methyl malonyl CoA to succinyl CoA. A defi-
ciency leads to pernicious anemia, at one time a
disease whose prognosis was death. Because fo-
lic acid can mask the blood picture of a cobal-
amin deficiency, it has become important for
physicians to order tests specific for the vitamin.
3.1 2.1 Chemistry. The cobalamin family
consists of a corrin ring (Fig. 8.54). It is similar
to that of the porphyrin ring system, except
that there is no methylene or methine bridge
between pyrrole rings A and D, and it contains
cobalt rather than iron. The commercial form
sold in the United States is cyanocobalamin.
The hydroxy dosage form also has been used.
The coenzyme forms include methyl and ad-
enosyl cobalarnin. The commercial vitamin is
produced from bacterial fermentation.
3.12.2 Uptake. Uptake of the vitamin
from food and vitamin products is complex.
Indeed, most deficiencies are not from inade-
quate diet, but result from defects in the up-
take process. Dietary cobalamin requires a
DHF (Dihydrofolate)
DHF * Folic Acid (standard
DHF Reductase mOnoglutarnate dosage
form)
fully functioning stomach and ileum of the
small intestine (73).Parietal cells in the stom-
ach produce hydrochloric acid, to free the vi- .
tamin from the food, and "intrinsic factor," a
glycoprotein that binds cobalamin. This com-
plex attaches to specific receptors in the ileum.
Eventually, the vitamin passes into systemic
circulation and is transported by a series of
plasma-binding proteins, the transcobal-
amins. About 50% of the absorbed vitamin
reaches the liver, with the remainder trans-
ported to other tissues.
Humans are efficient recyclers of vitamin
B,, because of enterohepatic circulation. The
vitamin is secreted in the bile. Upon combin-
ing with intrinsic factor, the absorption pro-
cess is repeated. This recirculation probably
explains why dietary deficiencies are uncom-
mon and why the inability to produce intrinsic
factor results in vitamin B,, deficiency, even
though there may be adequate dietary intake.
3.12.3 Biochemical Role. Cobalamin is a
coenzyme in only two reactions, but they are
basic to the health of the individual. Methyl
 /
H2C
/
0

O=P 0 OH
I
0- O
.
R = -CN, -OH, CH3,

OH OH
Figure 8.54. V i t a m i n B,, and coen-
zyme forms. Adenosyl

cobalamin is required for the regeneration of the rearrangement of methylmalonyl CoA to

methionine from homocysteine. 5-Methyltet- succinyl CoA with adenosylcobalamin as the
rahydrofolate polyglutarnate is also required coenzyme (Fig. 8.55). Odd-numbered fatty ac-
(Figs. 8.49 and 8.53). The second reaction is ids and the amino acids d i n e , isoleucine, and

0
\\
H3C-CH
f-scOA
\ 7
Biotin
H3C-CH
rsc
I
4 co2 b- O-
Propionyl CoA

Methylmalonyl-
CoA epimerase
v
0 0
A' \\
/"-" Adenosyl
cobalamin
- O-
H2C-CH H3C-CH
COAS 3 \H Methylmalonyl-
COA mutase )--SC A O
0 0
Figure 8.55. Cobalamin-catalyzed mu-
tase reaction. Succinyl CoA L-Methylmalony\CoA
3 Vitamins

methionine produce propionyl CoA as part of 3.1 2.6 Dietary Reference Intakes.
their catabolism. Biotin-catalyzed carboxyla- AT
tion (Fig. 8.45) yields D-methylmdonyl CoA, Infants 0.4-0.5 pglday
which must be epimerized to the L-stereoiso- EAR
mer. The latter undergoes a rearrangement to Children (1-13 years) 0.7-1.5 &day
succinyl CoA, which enters the Kreb cycle for Adolescents (14-18 years) 2.0 pglday
final degradation. There has been consider- Men and Women 2 pglday
able debate as to the mechanism of this rear- (19-50+ years)
rangement, but the general consensus is that Pregnancy 2.2 pglday
there are free-radical intermediates (74-76). Lactation 2.4 pglday
RDA
3.12.4 Cobalmin Deficiency. Pernicious Children (1-13 years) 0.9-1.8 pg/day
anemia is the disease associated with vitamin Adolescents (14-18 years) 2.4 d d a y
B,, deficiency. It is usually caused by the in- Men and Women 2.4 pglday
ability to produce intrinsic factor. Indeed, (19-50+ years)
many times the vitamin must be administered Pregnancy 2.6 pglday
by injection. The blood picture, a megaloblas- Lactation 2.8 pglday
tic anemia, is indistinguishable from that UL
caused by folic acid deficiency. Indeed folic None reported
acid supplements can mask the blood picture.
This is illustrated in Fig. 8.53. Removal of ad- 3.1 3 Ascorbic Acid (Vitamin C) (77)
enosyl cobalamin eliminates the regeneration
of tetrahydrofolate during the methylation of Deficiencies of this vitamin have been associ-
homocysteine to methionine. Folic acid sup- ated with the early sailors who lacked fresh
plements provide a fresh source of tetrahy- fruit and vegetables. However, it was a prob-
drofolate coenzymes. DNA synthesis can lem on land and was seen in the Irish potato
continue and new erythrocytes form. Excess famine, the California gold miners, and terri-
folic acid also may compete for the available torial prisons. There long has been a mystique
vitamin, further exacerbating vitamin B,, surrounding this vitamin, with interest
deficiency. sharply increasing when Linus Pauling pub-
Pernicious anemia can be lethal if not lished his book Vitamin C and the Common
treated because of nerve damage. There are Cold, forcing the medical, nutritional, and bio-
two explanations for the cause of this damage, chemical professions to reexamine carefully
both involving an excess of methylmalonyl the role of this essential nutrient in human
CoA. Methylmalonyl CoA is a competitive in- health. A significant problem with studying
hibitor of malonyl CoA during fatty acid syn- this vitamin is the fact that ascorbic acid is not
thesis. This may impede repair of the myelin a vitamin in most animals. It was not until the
sheath surrounding nerves. Alternatively, discovery that guinea pigs also require ascor-
methylmalonyl CoA replaces malonyl CoA as a bate that animal experiments could be con-
substrate in fatty acid synthesis, producing ducted.
fatty acids with methyl substituents. These
are incorporated into the lipids components of 3.1 3.1 Chemistry. Ascorbic acid is derived
the myelin sheath, producing a nonfunction- from the aldonic acid form of L-gulose (Figs.
ing myelin sheath. 8.56 and 8.57). There are two enolic proton-
donor groups, with the one at position 3 being
3.1 2.5 Hypervitaminosis B, ,. The vitamin the most acidic with a pK, value of 4.1. Ascor-
is considered nontoxic. There has been some bic acid is easily oxidized to the dehydro form
concern that the presence of the CN- anion in without loss of vitamin activity, but the lac-
the commercial vitamin might cause problems tone ring now hydrolyzes easily, producing in-
with megadoses. However, 1000 pg of cyan- active open chain product.
ocobalamin contains only 0.02 mg of CN. Most animals, except for primates and
There are no Tolerable Upper Intake Levels. guinea pigs, produce their own ascorbic acid
 Vitamins

\
pKa 4.2 HO' OH pKa 11.6

L-Ascorbic acid Dehydro-L-Ascorbic acid Sodium L-Ascorbate

Figure 8.56. Ascorbic acid chemistry.

HO- CH
I

k boH
OH
CH20H
I

-..;-
OH
OH - --.-----+
several
Steps
6

OH
co2-
I

OH
Glucuronate

NADPH + H+ NADP'
HO- CH
I
HC-OH
I
HO-CH
I
1 CH20H
D-Glucose D-Glucuronate L-Gulonic acid

Aldono-
lactonase
H20

I H. O, I
HO
/'-cH
\
yo 12 HI
\ HO
,c:AO)-o
\

HO
/"= "\
OH
Gulonolactone
oxidase
HO
pH.cH
\
OH
L-Ascorbic acid L-Gulonolactone

Figure 8.57. Outline of ascorbic acid biosynthesis.

3 Vitamins

Table 8.5 Metabolic Roles of Ascorbic Acid (Vitamin C)

Enzyme
Peptidyl-glycine monooxygenase
4-Hydroxyphenyl-
pyruvatedioxygenase
Proline hydroxylase
Lysine hydroxylase
Trimethyl lysine hydroxylase
4-Butyrobetaine hydroxylase
Cytochrome P450 isozymes
Reaction
Hydroxylate dopamine
phenethyl chain
Amidate carboxyl end of peptide
hormones
Hydroxylate phenylalanine
Posttranslational Hydroxylation
of proline
Posttranslational hydroxylation
of lysine
Hydroxylation of trimethyl
lysine
Oxidation of 4-butyrobetaine
aldehyde
Oxidation of steroids
Contribution
Synthesis of norephrine
Biosynthesis of peptide
hormones
Synthesis of tyrosine
Crosslinking of collagen
Crosslinking of collagen
Carnitine synthesis
Carnitine synthesis
Corticosteroid biosynthesis

from glucose (Fig. 8.57). The pathway follows
the standard route to glucuronic acid. The al-
dose carbon is reduced to an alcohol and, fol-
lowing normal carbohydrate-naming conven-
tion, the former carbon 6 of D-glucuronic acid
becomes carbon 1 of L-gulonic acid. Cyclic
L-gulonolactone forms and is oxidized to
L-ascorbic acid. Humans and primates lack gu-
lonolactone oxidase.
3.13.2 Uptake. Ascorbic acid is absorbed
from the intestine by a sodium-dependent ac-
tive transport system that is saturable. As the
concentration of vitamin C increases in the
intestinal tract, the absorption changes to pas-
sive diffusion. Once in systemic circulation,
there are specific transporters based on cell
types.
3.1 3.3 Metabolic Roles. Ascorbic acid is an
electron donor required for a variety of oxida-
tive processes. It is readily regenerated by glu-
tathione, NAD, and NADP and thus has a long
biological half-life. Currently, there are eight
known human enzymes that require ascorbic
acid, and they are listed in Table 8.5. The pre-
cise metabolic roles have not been completely
elucidated, but it appears that in the met-
alloenzymes, ascorbate reduces the active
metal site. In addition to these specific en-
zymes, ascorbic acid seems to function as a
free-radical scavenger in the aqueous phase of
plasma and cells.
3.1 3.4 Ascorbic Acid Deficiency. Scurvy is
the classical disease associated with ascorbate
deficiency. It is a disease of the connective tis-
sue and probably is caused by inadequate
crosslinking attributed to a lack of hydroxy-
lated proline and lysine. Many consider scurvy
to be an advanced stage of ascorbate defi-
ciency. Chronic deficiencies may also (1)in-
crease risk for malignancies, as evidenced by
oxidized DNA markers and increased concen-
trations of reactive oxygen species; (2) de-
creased immune function, as evidenced by less
vitamin in neutrophils and lymphocytes; (3)
cardiovascular disease caused by the inflam-
matory response on the blood vessel walls; and
(4) cataract formation caused by decreased
concentrations of ascorbate in the ocular tis-
sues.
3.1 3.5 Hypervitaminosis C. The vitamin is
considered very safe. At one time, many of
the over-the-counter products contained sig-
nificant amounts of sodium ascorbate, which
would be contraindicated in people on low
sodium diets. Today's products are virtually
sodium free unless labeled otherwise. Nev-
ertheless, there are intermittent reports of
adverse reactions associated with high
doses. Therefore, there are Tolerable Upper
Intake Levels, but these are very high rela-
tive to the RDAs. The UL to RDA ratio aver-
ages about 20.
 Vitamins

3.1 3.6 Dietary Reference Intakes. 6. Dietary Reference Intakes for Vitamin C, Vita-
min E, Selenium and Carotenoids, Food and
A1 Nutrition Board, Institute of Medicine, Na-
Infants 40-50 mglday tional Academy Press, Washington, DC, 2000.
EAR 7. Dietary Reference Intakes for Calcium, Phos-
Children (1-8 years) 13-22 mglday phorus, Magnesium, Vitamin D and Fluoride,
Boys (9-18 years) 39-63 mglday Food and Nutrition Board, Institute of Medi-
Girls (9-18 years) 39-56 mglday cine, National Academy Press, Washington, DC,
Men (19-70+ years) 75 mglday 1997.
Women (19-70+) 60 mglday 8. Dietary Reference Intakes for Thiamin, Ribofla-
Pregnancy 66-70 mglday vin, Niacin, Vitamin B , Folate, Vitamin BIB
Lactation 96-100 mglday Pantothenic Acid, Biotin and Choline, Food and
RDA Nutrition Board, Institute of Medicine, Na-
15-25 mglday tional Academy Press, Washington, DC, 1998.
Children (1-8 years)
Boys (9-18 years) 45-75 mglday 9. See Ref. 5, pp. 82-131.
Girls (9-18 years) 45-65 mglday 10. C. E. West, Nutr. Rev., 58,341-345 (2000).
Men (19-70+ years) 90 mglday 11. R. Blomhoff, M. H. Green, T. Berg, and K. R.
Women (19-70+) 75 mglday Norum, Science, 250,399-404 (1990).
Pregnancy 80-85 mglday 12. Information on approved drugs in this chapter
115-120 mgtday will be found in the package inserts. These are
Lactation
available in the Physicians Desk Reference, USP
UL Drug Information for the Health Care Profes-
Infants Not established; sional, and the Food and Drug Administration
use formula web site, www.fda.gov.
and food only 13. L. Roberts, Science, 239,564 (1998).
Children (1-8 years) 400-650 mglday 14. M. S. Tallman, N. Engl. J. Med., 337, 1021-
Boys and Girls (9- 1200 mglday 1028 (1997).
13 years) 15. See Ref. 7, pp. 250-257.
Adolescents (14-18 1800 mglday 16. W. F. Loomis, Sci. Am., 223,76-82 (1970).
years) 17. M. T. Weick, Am. J.Clin. Nutr., 20, 1234-1241
Adults (19+ years) 2000 mglday (1967).
Pregnancy 1800-2000 mglday 18. J. A. MacLaughlin, R. R. Anderson, and 6I.F.
Lactation 1800-2000 mglday Holick, Science, 216, 1001-1003 (1982).
19. K. T. Koshy, J.Pharm. Sci., 71,137-153 (1982).
20. P. P. Minghetti and A. W. Norman, FASEB J.,
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BIBLIOGRAPHY A N D FURTHER READINGS
Dietary Reference Intakes for Calcium, Phospho-
rus, Magnesium, Vitamin D and Fluoride,
Food and Nutrition Board, Institute o f Medi-
cine, National Academy Press, Washington,
DC, 1997.
Dietary Reference Intakes for Thiamin, Riboflavin,
Niacin, Vitamin B , Folate, Vitamin B , , Panto-
thenic Acid, Biotin and Choline, Food and Nutri-
Vitamins
tion Board, Institute o f Medicine, National Acad-
emy Press, Washington, DC, 1998.
Dietary Reference Intakes for Vitamin C, Vitamin E,
Selenium and Carotenoids, Food and Nutrition
Board, Institute o f Medicine, National Academy
Press, Washington, DC, 2000.
Dietary Reference Intakes for Vitamin A, Vitamin K,
Arsenic, Boron, Chromium, Copper, Iodine, Iron,
Manganese, Molybdenum, Nickel, Silicon, Vana-
dium and Zinc, Food and Nutrition Board, Insti-
tute o f Medicine, National Academy Press,
Washington, DC, 2001.
CHAPTER NINE

Lifestyle and Over-the-Counter

Drugs
K H A m ABU-IZZA
Sanofi-Synthelabo Research
Malvern, Pennsylvania

VINCENT LI
Wyeth Consumer Healthcare
Richmond, Virginia

GRAHAM PARR
Wyeth Consumer Healthcare
Hampshire, United Kingdom

Contents
1 Introduction, 422
2 Self-Medication, 423
3 Over-the-counter Drugs, 423
3.1 OTC Drug Classification
in the United States, 424
3.1.1 History of the Regulatory Approval
Process, 424
3.1.2 OTC Drug Review, 424
3.1.2.1 Phase 1: Advisory Panel
Review, 424
3.1.2.2 Phase 2: FDA Review, 425
3.1.2.3 Phase 3: The Final Monograph,
425
3.1.3 OTC Classification After OTC Drug
Review, 426
3.1.4 Criteria for Reclassification of Drug/
Rx-to-OTC Switch, 427
3.1.4.1 Safety, 427
3.1.4.2 Effectiveness, 427
3.1.4.3 Labeling, 427
3.2 OTC Classification in Europe, 428
3.3 OTC Classification in Japan, 430
4 Lifestyle Drugs, 431
5 Hair Growth Disorders, 433
Burger's Medicinal Chemistry and Drug Discovery 5.1 Clinical Use, 433
Sixth Edition, Volume 4: Autocoids, Diagnostics, 5.1.1 Current Drugs, 433
and Drugs from New Biology 5.1.2 Adverse Effects, 434
Edited by Donald J. Abraham 5.1.3 Pharmacokinetics, 434
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc. 5.2 Physiology and Pharmacology, 435
422
5.2.1Physiology of Hair Growth, 435
5.2.2Role of 5-a-Reductase, 435
5.2.3Mechanisms of Action, 435
5.2.3.1 Minoxidil, 435
5.2.3.2 Finasteride, 436
5.2.3.3Eflornithine, 436
5.3Chemistry and Structure-Activity
Relationships, 437
5.3.1Minoxidil, 437
5.3.25a-Reductase Inhibitors, 437
5.4History, 438
5.5Current and Future Trends, 440
6 S e u a l Disorders, 440
6.1Clinical Use, 442
6.1.1Current Drugs, 442
6.1.2Adverse Effects, 443
6.1.3Pharmacokinetics, 444
6.1.3.1Sildendl, 444
6.1.3.2Alprostadil, 444
6.1.3.3Yohimbine, 444
6.2Physiology and Pharmacology, 444
6.2.1Physiology of Erection, 444
6.2.2Role of Nitric Oxide, 445
6.2.3Other Mediators, 445
6.2.4Causes of Erectile Dysfunction, 445
6.2.5Mechanisms of Action, 446
6.2.5.1Sildendl, 446
6.2.5.2Alprostadil,446
6.2.5.3Yohimbine, 446
6.2.5.4Other Agents, 446
6.3Chemistry and Structure-Activity
Relationships, 446
6.4History, 447
6.5Future Trends, 449
7 Smoking Cessation Agents, 450
7.1Clinical Use, 451
7.1.1Current Drugs, 451
7.1.2Adverse Effects, 451
7.1.3Pharmacokinetics, 453
7.2Physiology and Pharmacology, 454
7.2.1Pharmacological Action of Nicotine,
454
1 INTRODUCTION
The landscape of the pharmaceutical industry
has undergone significant changes in recent
years. The changes were brought about by in-
creased demand from consumers to take con-
trol of their health (1-3), direct-to-consumer
(DTC)advertising of prescription drugs (4,5),
technological advances in genomic research
(6), and the search for blockbuster drugs by
pharmaceutical companies (7). Today, con-
Lifestyle and Over-the-counter Drugs
7.2.1.1Neurological Basis of Nicotine
Dependence, 454
7.2.1.2Peripheral Pharmacological
Actions of Nicotine, 455
7.2.2Bupropion Mechanism of Action, 455
7.3Chemistry and Structure-Activity
Relationships, 455
7.3.1Nicotinic Acetylcholine Receptors, 455
7.3.2Structural Requirements for nAChR
Ligands, 456
7.3.3SAR of Bupropion and Its Analogs, 456
7.4History, 457
7.5Current and Future Trends, 458
8 Sunscreens, 458
8.1Clinical Use, 460
8.1.1Current Drugs, 460
8.1.2Side Effects, 461
8.1.3Absorption and Disposition, 462
8.2Physiology and Pharmacology, 463
8.2.1Acute Effects of UVR, 464
8.2.2Chronic Effects of UVR, 464
8.2.2.1Adaptive Responses, 464
8.2.2.2Photoaging, 465
8.2.2.3Carcinogenesis, 465
8.2.3Mechanisms of Action of Sunscreens,
466
8.2.4Sun Protection Factor, 467
8.3Chemistry and Structure-Activity
Relationships, 467
8.3.1Aminobenzoates, 467
8.3.2Salicylates,468
8.3.3Cinnamates, 469
8.3.4Benzophenones, 469
8.3.5Anthranilates, 469
8.3.6Dibenzoylmethanes, 469
8.3.7Physical (Particulate)Sunscreens,470
8.4History, 470
8.4.1Emergence of Sunscreens, 470
8.4.2History of FDA Regulations,470
8.5Current and Future Trends, 472
9 Future Trends for OTC and Lifestyle Drugs, 473
sumers are more informed and want to take
control of their health. The best example is the
increased popularity of dietary supplements
and the consumer lobbying to pass the Dietary
Supplements Education and Health Act in the
United States (8, 9). The relaxation of the di-
rect-to-consumer advertising regulation in
1997 provides an excellent avenue for the
pharmaceutical industry to promote new ther-
apeutic categories of prescription drugs such
as lifestyle drugs that cater to lifestyle en-
3 Over-the-counter Drugs
hancement to the consumers. The desire of
the aging baby-boomer to maintain an active
lifestyle (10) and the difficulty of searching for
blockbuster drugs in traditional therapeutic
areas provide an excellent environment for
the birth of lifestyle drugs.
The landscape of self-care has also under-
gone significant changes in recent years as
consumers become more informed and as
more highly effective prescription drugs (Rx)
are switched to over-the-counter (OTC). The
demand of the baby-boomer consumers to look
and feel young has given rise to the popularity
of dietary supplements (13, 14) and the so-
called "cosmeceuticals" (10, 11).Some of the
more successful Rx-to-OTC switches (e.g.,
hair-growth, smoking cessation) in recent
years are related to lifestyle enhancement.
Such lifestyle-enhancing products help create
new therapeutic areas in the OTC drug arena.
This chapter will attempt to show how life-
style drugs and over-the counter-drugs relate
to each other and the challenges that they
face. At first glance, they seem to be oceans
apart; the former are mainly available by pre-
.
scriptions and the latter are available without
prescription. However, lifestyle drugs bear
some resemblance to the over-the-counter
drugs in that both are primarily for non-life
threatening conditions and are heavily pro-
moted by direct-to-consumer advertisement.
Moreover, consumers and patients are taking
an active role in choosing these two groups of
products. Furthermore, they both cater to im-
prove the quality of life of the consumers. Fi-
nally, lifestyle prescription drugs with low side
effects will be ideal candidates for Rx-to-OTC
switch. The following sections will provide an
overview of OTC drugs, selected examples of
lifestyle drugs, and the future of both groups.
2 SELF-MEDICATION
Before we discuss OTC drugs and lifestyle
drugs, we would like to provide a brief over-
view of self-medication to help gain an appre-
ciation of the changing attitudes of consum-
ers. A recent survey conducted by Roper
Starch Worldwide Inc. on behalf of the Con-
sumer Healthcare Products Association
(CHPA) showed that American consumers are
increasingly comfortable with self-medication
for minor medical problems. Some of the find-
ings (2) are as follows:
0 73% would rather self-treat than see a doc-
tor
0 96% say they are confident about their
healthcare decision
0 80% used at least one OTC product to treat
minor ailments
0 approximately 90% of consumers read the
label for possible side effects the first time
they use an OTC product
0 57% of consumers say they are using or con-
sidering the use of dietary supplements
0 80% of those who take dietary supplements
are happy with the outcomes
The above attitudes toward self-medication
strongly suggest that American consumers
cherish self-reliance and convenience. Similar
attitudes are held by European consumers (3,
15) and are beginning to shape Japanese con-
sumers (16). Today, consumers are generally
better informed because of DTC advertising
on television and the vast amount of medical
information available on the internet. Con-
sumers use OTC products to treat a variety of
minor ailments such as headache, upset stom-
ach, cough, and cold.
3 OVER-THE-COUNTER DRUGS
In the United States, there are two classes of
drug products: prescription and nonprescrip-
tion. Nonprescription drugs are often referred
to as over-the-counter drugs because they are
available for general sale to consumers with-
out a prescription (17). Consumers can pur-
chase OTC drug products off the shelf from a
variety of mass distribution outlets such as
convenience, grocery, and drug stores, with
the exception of Schedule V Controlled Sub-
stance and insulin products, which are kept
behind the pharmacist counter and dispensed
by pharmacists. In European countries, most
OTC drugs are only available at the pharmacy,
although in some countries, e.g., U.K., the sirn-
plest products are widely available through gen-

eral sale (18, 19). However, there is a growing
trend to allow more non-prescription products
to be sold outside the pharmacy. When we
speak about OTC products, we also include
medical devices such as pregnancy test kits
and dietary supplements. This chapter will fo-
cus on OTC drugs only. The emphasis will be
on the U.S. OTC drug classification process,
with a brief overview of the OTC drug classi-
fication processes in Europe and Japan.
3.1 OTC Drug Classification
in the United States
To gain a better understanding of the OTC
classification process, we need to take a look at
the history of the regulatory process in the
United States.
3.1.1 History of the Regulatory Approval
Process. The process of drug approval under-
went an evolutionary change throughout the
20th century (20, 21). Major changes are in-
variably triggered by some public health haz-
ards. The first major piece of legislation at the
beginning of this century was the 1906 Pure
Food and Drug Act, which mandated that the
labeling on the package be truthful. There was
no requirement for safety and efficacy as we
know today. The New Drug Application (NDA)
process did not begin until the passage of the
original 1938 Federal Food, Drug and Cos-
metic (FD&C)Act, which only demanded that
the manufacturer submitted an NDA with
safety data before marketing a new drug. The
manufacturer could market the product if the
FDA had no objection to the NDA within 60
days. It was up to the manufacturer to decide
whether or not the new drug would be pre-
scription or OTC. The distinction between
prescription drugs and OTC drugs is based on
the Durham-Humphrey amendment in 1951,
which limited the following three classes of
drugs to prescription status (22):
1. Certain habit-forming drugs
2. Drugs that cannot be safely used without
professional supervision
3. Drugs limited to prescription under an NDA
A subsequent 1962 amendment (Kefauver-
Harris) to the 1938 FD&CAct in 1962 clarified
Lifestyle and Over-the-Counter Drugs
that drug products must be both safe and effi-
cacious and be approved by the FDA before
marketing (23). A s a result of the 1962 amend-
ment, the FDA was required to retrospectively
review all NDAs approved between 1938 and
1962 for both efficacy and safety. The FDA, in
collaboration with the National Academy of
Sciences and National Science Foundation, re-
viewed all NDAs submitted between 1938 and
1962 through the Drug Efficacy Study Imple-
mentation (DESI) review program (22-25).
This review focused primarily on prescription
products. After the completion of the review of
the prescription products, FDA turned their
attention to OTC products.
3.1.2 OTC Drug Review. In the 1960s, the
OTC drug market back then was similar to to-
day's market in that there was a proliferation of
products based on the same actives (24-28). For
this reason, it became impossible to review every
OTC product on the market. For practical rea-
sons, the FDA decided to review active ingredi-
ents by therapeutic categories instead of individ-
ual finished products. In 1972, the FDA initiated
the OTC Drug M e w process. The goal of the
OTC Drug Review was to determine which ac-
tive ingredients and their associated conditions
of use might be considered to be generally re-
garded as safe (GRAS) and efficacious (GRAE)
and not misbranded. The FDA set up a total of
17 public advisory panels to review close to 90
categories of OTC drugs (24,25). All OTC prod-
ucts, including Grandfather OTC products in-
troduced before 1938, were also reviewed. The
review was conducted in three rule-making
phases.
3.1.2.1 Phase 1: Advisory Panel Review.
The panels were asked to categorize the active
ingredients in each class of products and their
claims into one of three categories based on
review of data submitted by manufacturers,
scientific data in the literature, and their own
experiences. The three categories (24,251 are
as follows:
0 Category I: generally recognized to be safe
and effective for the claimed indications
m Category 11: not generally recognized as safe
and effective or unacceptable for the
claimed indications
3 Over-the-counter Drugs
Table 9.1 OTC Monographs
Already Published
Acne
Anorectal
Antacid
Anthelmintic
Antibiotic first aid
Anticaries
Antiemetic
Antiflatulent
Antifungal
Cholecystokinetic
Corn/callus removers
Coughlcold antihistamine
Coughlcold antitussive
Coughlcold bronchodilator
Coughlcold expectorant
Coughlcold nasal decongestant
Dandruff/seborrhedpsoriasis
Deodorants (internal)
Male genital desensitizers
Nighttime sleep aids
Otic (earwax)
Pediculicide
Stimulant
Sunscreen
Wart remover
Weight control
Source: Ref. 28 (Reproduced with permission from Consumer Healthcare Products As-
sociation).
Category 111: insufficient data available to
permit final classification into Category I or
Category I1
The above three-category classification sys-
tem is not in use today. Drugs applied for OTC
use are classified either as "monograph" or
"non-monograph" status. The findings of the
advisory panels were published in the Federal
Registry as "advance notice of proposed rule-
making" (ANPR), which sets forth the advi-
sory panel's recommendation for OTC mono-
graph by category of use (Table 9.11, including
recommendations on general recognition of
safety and effectiveness for each active ingre-
dient. This phase of review took about 10
years to complete.
3.1.2.2 Phase 2: FDA Review. After publi-
cation of an advance notice of proposed rule-
making, any interested party may submit
comments and additional new data to support
their point of view. The FDA reviews all the
Still Pending
Antidiarrheal
Antiseptic first aid
Antiseptic (professional)
Antiperspirants
Coughlcold combinations
External analgesic
Internal analgesic
Laxative
Menstrual
Oral discomfort
Oral health care
Otic (ear drying)
Overindulgence
Poison treatment
Skin bleaching- (reproposal)
Skin protectants
Vaginal drug products (douches)
comments and newly submitted data and pub-
lished their rulings in a "tentative final mono-
graph" (TFM) or proposed rule, which sets
forth FDA's revisions to the ANPR relating to
active ingredients and conditions of use. The
TFM groups actives together for specific indi-
cations to form what we now call a "mono-
graph" (Table 9.1). The rulings on those ac-
tives and indications where no claims can be
made are called "negative monographs" (Ta-
ble 9.2). In a similar manner to the ANPR,
additional comments and data could be sub-
mitted to clarify or refute the rulings.
3.1.2.3 Phase 3: The Final Monograph. After
reviewing comments to the proposed rule (or
TFM), the FDA publishes the "Final Mono-
graph" in the form of a "Final Rule" in the
Federal Register, which is then incorporated
into the Code of Federal Regulations (21CFR
part 330). The final monograph consists of a
list of active ingredients approved for the spe-
cific indications, labeling, general provisions,

Table 9.2 Negative Monograph (Non-Monograh)
Aphrodisiac
Benign prostatic hypertrophy
Boil treatment
C/C anticholinergic
Daytime sedatives
Digestive aids
Exocrine pancreatic insufficiency
Fever blisterlcold sore (internal)
Hair grower
Source: Ref. 28 (Reproduced with permission from Consumer Healthcare Products As-
sociation).
and testing procedures. During the OTC Drug
Review, misbranded products that did not
meet the monograph requirements would
need to be reformulated, or their labels needed
to be revised to stay on the market. A drug
manufacturer can market an OTC product
based on those active ingredients for the in-
tended indications described in the mono-
graph without the need of an NDA submis-
sion. However, a drug manufacturer cannot
make claims not described in the monograph
unless they submit additional data to petition
for a revision of the final monograph or justify
their deviations and obtain approval through
an NDA. Even though the monographs are
based on active ingredients and not on the dos-
age forms, the implicit assumption is that the
dosage forms (e.g., tablet, capsule, liquid) will
not affect the efficacy and safety of the active.
Whereas acetaminophen immediate-release
tablets, capsules, and liquids can be marketed
without an NDA submission, marketing a sus-
tained-release acetaminophen tablet will still
require a submission of an NDA deviation.
Although most of the final monographs have
been published, there are still 17 final mono-
graphs awaiting completion (Table 9.1). The
OTC review process is dynamic in the sense that
final monographs can be amended. New ingre-
dients or indications can also be petitioned for
monograph status based on material time and
material extent (29). For example, foreign OTC
products and existing OTC actives approved
through an NDA can be petitioned for mono-
graph status after a certain period of marketing.
3.1.3 OTC Classification After OTC Drug
Review. The OTC Drug Review is based on
the principle set down in the Durham-Hum-
Lifestyle and Over-the-counter Drugs
Hypohyperphosphatemia
Ingrown toenail
Insect repellant (oral)
Nail bitinglthumbsucking
Oral wound healing
Prevention of inebriation
Smoking deterrents
Stomach acidifiers
Vaginal contraceptives
phrey amendment that if a drug can be OTC, it
should be OTC (23, 30). Prescription status
should be an exception. However, the history
of drug approval suggests otherwise. Most
new chemical entities are initially approved as
prescription drugs, which may then be
switched after a period of at least 5 years of
marketing experience and meeting certain
switch criteria (29). Direct OTC approval of
new chemical entity is a rare phenomenon.
Close to 30 drugs were reclassified from
prescription to OTC status during the OTC
Drug Review (24). Some of the common OTC
cough cold actives such as diphenhydramine,
brompheniramine, chlorpheniramine, and
pseudoephedrine, together with hydrocorti-
sone, were switched during that time. The
switch can go in both directions. Meta-
proterenol sulfate inhaler was switched by
FDA during the OTC Drug review and was
later reversed back to prescription status be-
cause of the objection of the medical commu-
nity. Today Rx-to-OTC switch is achieved by
submitting an NDA for the same indication or
new indications or new dose. Three-year mar-
ket exclusivity will be granted to the sponsor if
new clinical studies that are "essential" to the
switch of an existing prescription product are
conducted (31, 32). All the H, blockers that
were switched received this type of exclusivity
because additional studies were conducted to
support the heartburn prevention claims. Five
years exclusivity will be granted for a new
OTC chemical entity that has not been mar-
keted previously. A supplemental NDA is filed
if a direct switch is desired without additional
clinical studies. In this case, there is no mar-
keting exclusivity other than what may al-
ready exist through patent protection. The
3 Over-the-counter Drugs
first generation vaginal antifungal products
and minoxidil products represented direct
switches and received no exclusivity. With or
without exclusivity, successful Rx-to-OTC
switches represent major growth opportuni-
ties for OTC companies.
3.1.4 Criteria for Reclassification of Drug/
Rx-to-OTC Switch. The terms reclassification
and Rx-to-OTC switch can be used inter-
changeably (25, 27, 30-37). There are many
drivers for Rx-to-OTC switch. Pharmaceutical
companies use Rx-to-OTC switch to increase
the lifespan and profitability of a prescription
drug that is coming off patent. Switch can also
expand the market by tapping into a new pa-
tient population that previously can not be
reached as a prescription product. An example
is Imodium (38). Healthcare providers and
governments who fund national healthcare in-
surance would like to advocate for the switch
for cost-containment. Each Rx-to-OTC switch
is unique and is evaluated on a weight-of-the
evidence of science and data. Rx-to-OTC
switch does not limit to self-limited, acute con-
ditions. The scientific/regulatory paradigms of
reclassification of drugs used by the OTC Drug
Review are still applicable to today's Rx-to-
OTC switches (30, 36, 37). Reclassification is
based on the principles of benefit-risk assess-
ment of safety and efficacy of the switched
drug (33). The switch criteria center around
the ability of a prescription product to meet
the safety, effectiveness, and labeling require-
ment of being an OTC self-care product for a
specific indication. Safety, effectiveness, and
labeling are the three most important criteria
in deciding potential Rx-to-OTC switches. The
following sections describe the requirements
of safety, effectiveness, and labeling for Rx-to-
OTC switches in more detail.
3.1.4.1 Safety. Safety is especially impor-
tant for OTC products because they are used
without professional supervision. Safety does
not mean that there is no potential toxicity
nor abuse potential (33). It does imply a low
incidence of adverse reactions or significant
side effects under adequate labeling and a low
abuse potential for harm (39). No drug is de-
void of side effects, but the margin of safety
should be much wider for OTC drugs com-
pared with that of prescription drugs. The
FDA demands that the drug manufacturer es-
tablishes a minimum effective dose and de-
fines a daily allowable dose to minimize undue
side effects. The switched Rx drugs should be
free of any carcinogenic potential in humans.
Reproductive toxicity, if any, should be ad-
dressed by proper OTC labeling. An example is
the pregnancylnursing warning for aspirin-
containing OTC products because of the po-
tential of Reyes syndrome (40). Any toxicity
observed in animals should also be shown to
be species-specific and have no relevance to
humans.
3.1.4.2 Effectiveness. FDA regulation de-
fines effectiveness as a reasonable expectation
that the drug will have the claimed therapeu-
tic effect in the targeted population when used
according to instructions in the label (41). Ef-
fectiveness is demonstrated by controlled clin-
ical studies. The clinical development for an
Rx-to-OTC switch program generally starts
with a dose ranging study to determine the
minimum dose to produce a therapeutic re-
sponse in most subjects in the target popula-
tion. Oftentimes, a lower dose is switched to
increase margins of safety. Actual use studies
are then performed in the target population to
demonstrate the effectiveness of the minimal
effective dose and identify any rare side effects
in OTC-like setting. For these reasons, the
number of patients involved can be quite sub-
stantial.
3.1.4.3 Labeling. Clear and understand-
able labeling to average consumers, including
those individuals with low literacy, is crucial
to the safe and effective use of an OTC product
without professional supervision. The label
should contain information such as active in-
gredient, indications, warnings, directions for
use, and proper storage condition. An average
consumer of the target population of users
should be able to understand the product label
to self-recognize the signs and symptoms, se-
lect the right OTC product, understand the
warnings, and self-treat appropriately as di-
rected (30). Warnings may include contraindi-
cations, precautions, situations to avoid, what
to do in the case of overdose, and drug-drug
interactions (42). However, not all warnings
are included in the label, only those that are
clinically significant and important to the safe
428
and effective use of the product by the con-
sumers. Labels should therefore be properly
design and tested.
Label comprehension studies are designed
to test the comprehensibility and readability
of the proposed label under general conditions
of purchase to ensure safe and effective use of
the OTC products. They become more impor-
tant for more complex Rx-to-OTC switches. In
general, the Rx-to-OTC switch sponsor will
work with FDA to define the comprehension
study objectives before conducting the study.
Several rounds of label comprehension studies
are performed to refine the comprehensibility
and readability of the label before initiating an
actual use study in the target population in a
simulated OTC setting to confirm the effec-
tiveness of the product using the finalized la-
bel. Oftentimes, a self-selection study is also
conducted to assess the ability of the con-
sumer to select or de-select the product. An
Investigational New Drug Application (IND)
is not always required for label comprehension
studies. An IND is required only if the sponsor
would like to discuss the studies with FDA.
The content of the label of a monograph
product should be consistent with the allow-
able indications and wordings specified by the
relevant monograph. For Rx-to-OTC switch
products, the label should be consistent with
the label approved in the NDA or supplemen-
tal NDA. To make labels easier for consumers
to read and comprehend, the FDA revised the
labeling requirements for OTC products in
1999 to standardize the content, format, font
size, headings, terminology and sequence of
information (43). The new OTC Drug Facts
label (44) contains six specific headings as de-
picted in Fig. 9.1 and resembles the Nutrition
Fact label of food and nutritional products.
3.2 OTC Classification in Europe
In Europe, the OTC regulatory landscape is
still fragmented despite the Mutual Recogni-
tion Procedure (MRP) and Directive 921261
EEC (18, 19, 45-47). Directive 92/26/EEC in-
tends to provide a uniform framework for the
classification of the medicinal products into
prescription and non-prescription and facili-
tates the switch process (46,47). However, its
application is left with the regulatory approval
agency of the individual country at the time
Lifestyle and Over-the-counter Drugs
when a marketing license is granted. This de-
centralized approach results in lack of consis-
tency from country to country. As a result,
some products are Rx in some countries but
are OTC in other countries (Table 9.3).
A closer look at Article 3 of Directive 921261
EEC revealed that a product is prescription if
it meets the following requirements.
1. It may present a danger if used without
medical supervision.
2. It could easily be misused or abused,
thereby causing harm to the consumers.
3. It causes side effects that require further
medical investigations.
4. It needs to be administered parenterally.
Article 4 of the Directive further stated
that medicinal products not classified as pre-
scription status should be non-prescription.
The principles for classification of medicinal
products in Directive 92/26 are very similar to
those in the United States. Despite the estab-
lishment of the Mutual Recognition Proce-
dure, there is as yet no unified interpretation
of the Directives. The interpretation of the Di-
rectives is left largely to the regulatory agency
of each country. For example, the United
Kingdom and Germany consider a switch with
reference to an active, whereas in the majority
of European countries, the switch is deter- I
mined on a product by product basis (18, 19). 1
Despite the lack of uniformity in the switch
process, most social insurance systems in Eu-
ropean countries are aggressively promoting
Rx-to-OTC switch as a means to contain
healthcare costs. The MCA in the U.K. has
already proposed a wide range of potential
switches in the next few years (48).
European countries do not have a Mono-
graph system to allow for rapid marketing of
OTC products. All OTC products require reg-
istration and marketing authorization before
marketing (46, 47). Marketing authorization
can be sought on a national level for market- j1
ing in just one country or using the Mutual
Recognization Procedure for marketing in
more than one country. The marketing autho-
rization is renewable every 5 years. When us-
ing the MRP, the company first files a market-
ing authorization application in a one of the
3 Over-the-counter Drugs

Drug Facts
Active ingredient Purpose
Name & amount per unit What is the product for,
e.g pain reliever, cough
suppressant ?
Uses
What symptom or diseases the product intend to treat?
Warnings (generally contain the following information)
r When not to use the product?
r Consult a doctor before taking the product.
r Possible side effects or drug-drug interactions
r When to stop taking the product and contact the doctor?
r If pregnant or breastfeed, ask a healthcare professional before use.
Keep out of reach of children
- - - -- --
Directions (generally contain the following information)
r Do not take more than directed
How much to take?
r Frequency
- & duration of treatment
r What age groups the product is for?
Other information (generally contains the following infonnation)
What is the proper storage condition?
Amount of calcium, potassium or sodium in the product
Inactive Ingredients (generally contain the following information)
Inactive ingredients in the product

Figure 9.1. An example of the new OTC Drug Facts label.

EU countries, which is designed as the refer-
ence member state (RMS). Once the RMS ap-
proves the product, it will then send an assess-
ment report to other EU member countries
the company desires to market the product.
The other EU countries are called the con-
cerned member states (CMSs). They will ap-
prove the product if they concur with the as-
sessment of the RMS. The CMS can disagree
with the assessment of the RMS based on
safety concerns. The company and the dissent-
ing CMS will go into arbitration. Oftentimes,
the many objections are political in nature,
making the MRP a rather protract and chaotic
process. Because the company cannot launch
the product in any CMSs unless the arbitra-
tion is resolved, they will generally withdraw
the application from the member country un-
dergoing arbitration to prevent undue delay.
The European Commission is considering
overhauling the MRP (47).
The non-prescription product landscape is
further complicated by the different modes of
distribution from country to country (49).
There are two major distribution channels for
non-prescription products in Europe; namely,
pharmacy only and general sale outlets, unlike
the United States, where OTC products are
for general sale only. For pharmacy-only prod-
ucts, some will need to be dispensed by the
pharmacist. Rx-to-OTC switch products are
usually placed in this category. The purpose of
pharmacy-only products is to provide profes-
sional consultation to consumers. However, a
430 Lifestyle and Over-the-counter Drugs

Table 9.3 Examples of Different Classifications of OTC Products Across Several European
Countries, Australia, and the United States
- - - -

U.S. Australia France Germany Italy Sweden Switzerland UK

Diclofenac Rx OTC OTC Rx OTC Rx OTC OTC
Ketoprofen OTC OTC OTC OTC OTC Rx Rx OTC
Clotrimazole (vaginal) OTC Rx OTC OTC Rx OTC Rx OTC
Loratadine Rx OTC Rx OTC Rx OTC Rx OTC
Acyclovir Rx OTC OTC OTC Rx Rx OTC OTC
Cimetidine OTC Rx OTC Rx OTC Rx Rx OTC
Farnotidine OTC Rx OTC Rx Rx OTC OTC OTC
Ranitidine OTC OTC OTC Rx Rx OTC OTC OTC
Nicotine (gum) OTC OTC OTC OTC OTC OTC OTC OTC
Nicotine (patch) OTC Rx OTC OTC OTC OTC Rx OTC
Source: Ref. 19.

study by the U.S. General Accounting Office
(GAO) did not find any differences of benefits
in either mode of distribution (49).
3.3 OTC Classification in Japan
Drugs in Japan are classified into prescription,
non-prescription (OTC), and quasi-drug. Pre-
scription drugs are those drugs that require
physician supervision (50-53). Non-prescrip-
tion drugs are primarily for mild actions and
are only available in the pharmacy or drug
store. Deregulation reform reclassified 15 cat-
egories of non-prescription drugs to create a
new class of drugs called quasi-drugs, which
can be sold in convenience stores and super-
markets starting in Spring 1999 (51). Quasi-
drugs are primarily used for external uses or
preventive purposes. They include the popular
tonic drinks, which are unique to the Japanese
OTC market.
All three classes of drug are regulated un-
der the Pharmaceutical Affairs Law adminis-
tered by the Ministry of Health and Welfare.
Prescription drugs and quasi-drugs are ap-
proved by Ministry of Health, Labor, and Wel-
fare (MHLW) only, whereas non-prescription
drugs are approved by both the MHLW and
local prefectural governments. OTC drugs are
approved by Evaluation Division I11 of Phar-
maceuticals and Medical Devices Evaluation
Center, which is under the MHLW. The coun-
try is administratively divided into 47 prefec-
tural governments (52). Registration applica-
tion submitted to MHLW is through the
prefectural government.
Similar to European countries, all OTC
products require regulatory approval (Shonin)
and a license (Kyoka) to market the product
(52). The approval time is generally longer
than in the United States. OTC drug applica-
tion is categorized into six classes (50,521. The
application system allows for both direct OTC
approval and Rx-to-OTC switch. Minoxidil
was switched directly to OTC status without
being a prescription product first. To expedite
and simplify OTC drug registration, the
MHLW transferred its approval authority to
prefectural governments (52). Specific mono-
graphs were established for each of these'cat-
egories.
Rx-to-OTC switch in Japan has for a long
time followed the principle-safety first and
efficacy second (53). For this reason, a lower
dose is often switched. Label comprehension is
not a big issue for Japanese OTC products be-
cause OTC products are sold primarily in the
pharmacy and a lot of graphics and cartoons
are used to communicate warnings and direc-
tions of use. Rx-to-OTC switches are usually of
low profile in Japan. The reason is that physi-
cians seldom support and advocate the switch.
Because physicians both prescribe and dis-
pense prescription drugs, the switch will de-
crease their business. There are a couple of
other reasons for poor performance of some of
the Rx-to-OTC switches. Consumers view
OTC products as less effective because of
lower dose and less safe because of warnings
listed on the label and in advertising. More-
over, co-pay for prescription drugs used to be
 4 Lifestyle Drugs

Table 9.4 A Few Definitions of Lifestyle Drugs

Source Definition
- - - - -

Gilbert et al. (63) "Drugs that are used to treat either non-health problems that are lifestyle wishes
and not medical necessities of health problems that might be better treated by
a change of lifestyle"
Herald Tribune (56) "Drugs that don't necessarily cure illness but can be used to improve daily life by
boosting psychological attitudes, energy levels, sexual performance and body
image."
Harth and Linse (66) "Pharmaceuticals which are taken in order to attain a certain psychosocial
beauty ideal rather than serve to stabilize the body's vital functions."
Business Week (54) "Drugs that are taken not for severely impaired diseases but for improving
quality of life."

less than that of OTC products. Furthermore,
no comparative advertising is allowed for OTC
products with approved standard mono-
graphs. However, Rx-to-OTC switch will be-
come more important in the future because
the government is trying to control healthcare
costs by increasing the co-pay.
4 LIFESTYLE DRUGS
Lifestyle drugs received much publicity (54-
56) since the launch of the little blue pill, Vi-
agra. The publicity is fueled partly by the hype
of the media and partly by the debates about
who is going to pay and what are their thera-
peutic benefits to the consumers (57-61). The
scientific field is equally interested in this class
of drugs, as reflected by a dedicated Scrip re-
port (62), an article in the British Medical
Journal (63), and a special series of articles in
the International Journal of Clinical Pharma-
cology (64-68).
Several factors, such as advances in tech-
nologies, growing consumerism, and demand
of baby-boomer consumers to look and feel
young, contribute to the popularity of lifestyle
drugs. Consumers want not only the absence
of disease but also improvement of quality of
life. Longer lifespans also give rise to a variety
of cosmetic and performance conditions that
E demand remedies.
! Several terms, such as vanity drugs, life-
1 enhancing drugs, and quality-of-life drugs, are
used by the media to describe lifestyle drugs
(69, 70). These are the terms pharmaceutical
companies try to dissociate themselves from
because they tend to project a rather negative
3 tone and create an image that lifestyle drugs
are to cater to desires rather than to treat real
medical problems. So exactly what are lifestyle
drugs? There are numerous definitions of life-
style drugs, depending on whom you talk to.
Table 9.4 lists a few definitions of lifestyle
drugs. The definitions range from attainment
of psychosocial beauty to treatment of health
problems that might be better treated by a
change of lifestyle.
The term "lifestyle drugs" refers to phar-
maceuticals, in particular prescription drugs,
that primarily aim to enhance the wellness
and improve the "desired" quality of life of an
individual rather than alleviating or curing
life-threatening diseases. Although all medi-
cations are used to improve the quality of life,
use of lifestyle drugs by consumers are in-
.
tended to attain a certain ideal quality of life
rather than to treat some life-threatening dis-
ease. The definition for lifestyle drugs may be
broadened to include drugs for the use of
treating serious medical conditions caused by
unhealthy lifestyles such as over-eating or for
off-label use, such as the use of Prozac, for
more self-confidence.
The focus of lifestyle drugs usually centers
around prescription drugs because of the issue
of who should pay for these medications and
their off-label use. The term lifestyle drugs
should not be limited to prescription drugs
only because some of them have already been
switched to OTC. Table 9.5 lists a few exam-
ples of lifestyle drugs and their indications.
These examples cover a broad range of drug
products. The range of indications is equally
broad; they range from preventive and cos-
metic to serious medical conditions such as
obesity and smoking.
432
Table 9.5 Examples of Lifestyle Drugs
Indications
Image and look
Wrinkles
Hair loss
Facial hair removal
Nail fungus
Increase muscle mass or height
Quality of life improvement
Incontinence
Impotence
Influenza
Lifestyle
Postpone menstruation
Smoking cessation
Contraception
Weight control
Dyspepsia
Sun protection
Psychological
Anti-depression
Obsessive-compulsive disorder
Social anxiety disorder
Source: Refs. 63, 69, 71.
Because the majority of lifestyle drugs are
not used to treat life-threatening diseases or
to alleviate pain, and the cost of coverage is
huge, the majority of debate has been focused
on who is going to pay (57,61). Most countries
have a national healthcare insurance system
that needs to control rising healthcare costs.
Coverage for lifestyle drugs puts considerable
strain on the limited resources of the insur-
ance system. The agency will therefore need to
rationalize the service and treatments. They
need to set priorities and determine which
treatments are medically useful and which
treatments are for lifestyle enhancement and
convenience only. The issue of what is medi-
cally needed is complicated by the heavy pro-
motion of lifestyle drugs by the pharmaceuti-
cal industries and transition of treatment
from specialists to general practitioners.
Without a doubt, the following scenario will
occur. Pharmaceutical industries will con-
tinue to push the envelop of medical innova-
tions and find ways to meet unmet consumer
lifestyle needs to increase their profitability.
The healthcare provider and national health
insurance system will continue to find ways to
control the cost of healthcare. The solution
Lifestyle and Over-the-counter Drugs
Drug
Tretinoin, vitamin A, botulium toxin type A, sunscreen
Minoxidil hasteride
Elornithine
Butenafine, terbinafine, itraconazole
Growth hormone
Oxybutynin, tolterodine
Sildefil, apomorphine
Zanamivir
Norethisterone
Nicotine replacement therapy, bupropion
Oral contraceptives
Orilstat, sibutramine, bupropion
Proton pump inhibitor
Sunscreens
Fluoxetine
Sertraline
Paroxetine
may be partially solved by switching the safer
lifestyle drugs to OTC because consumers are
willing to pay to obtain the benefit, and the
fact that they are already paying for many of
these lifestyle drugs.
The other controversy is what benefits do
lifestyle drugs offer the consumers because
they are primarily treating non-life-threaten-
ing diseases. Diseases such as incontinence
and impotency are not life-threatening, but
they create undue psychological stress on the
suffers. Furthermore, even for lifestyle drugs
that have legitimate therapeutic uses, they
can still be abused by healthy individuals for
purely performance enhancement. For life-
style drugs that are used to treat diseases
arisen from unhealthy lifestyles, drug cover-
age is only a partial and least-preferred solu-
tion. The best solution is for patients to take
control of their health by living a healthy life-
style. All these controversies will continue to
fuel the debate for coverage and benefits of
lifestyle drugs.
The following sections provide four exam-
ples of lifestyle drugs: hair growth compounds
for appearance enhancement, sexual disorder
compounds for performance remedy, smoking
5 Hair Growth Disorders

Table 9.6 Approved Treatments for Hair Growth Disorders

- -

Current
Brand Dosage Regulatory
Generic Name Name Originator Indication Form Daily Dose Status
Minoxidil(1) Rogaine Upjohn Androgenetic 2%,5% Topical OTC
alopecia solutions application
(men and twice daily
women)"
Finasteride (2) Propecia Merck Androgenetic 1 mg tablet 1 tabletlday Prescription
alopecia
(men only)
Eflornithine Vaniqa BMS Hirsutism 13.9% cream Topical Prescription
hydrochloride (women's application
(3) facial hair) twice daily
"The 5% is for men only.

cessation compounds for lifestyle change, and
sunscreens for lifestyle purposes. They span
the spectrum of OTC and prescription prod-
ucts.
5 HAIR GROWTH DISORDERS
Common baldness, also known as male-pat-
tern baldness, affects approximately 50% of
men by age 50 (72), although it can start as
early as the teen years. Till the late 1980s,
common baldness was viewed as a merely cos-
metic condition that has no health implica-
tions; with the introduction of hair growth
agents, the condition was medicalized and re-
defined as androgenetic alopecia. Androge-
netic alopecia affects women as well, but in a
different pattern that is usually referred to as
female-pattern baldness, diffuse hair loss, or
diffuse androgen-dependent alopecia. An esti-
mated 30% of Caucasian women, and 15-20%
of all women, develop the condition before age
50 (73, 74). Whereas hair loss, like other life-
style conditions, is not a direct threat to a per-
son's well-being, it can be a distressing and
psychologically disturbing condition because
of lower satisfaction with one's body image
(75, 76). In one of the clinical studies on mi-
noxidil, the majority of participants thought
that personal presentation of self was of equal
to or greater importance than their job perfor-
mance (77). Obviously, the condition has a
more severe psychological impact on female
subjects, which was confirmed in two separate
studies (78, 79).
Excessive hair growth in cosmetically un-
desirable areas is another hair growth disor-
der of little clinical importance but great life-
style impact. The condition, which is medically
known as idiopathic hirsutism, is defined as
increased hair growth in the androgen sensi-
tive areas of women with regular ovulatory
menstrual cycle and normal serum androgen
levels (80, 81). Hirsutism is a common condi-
tion that can have a severe psychological effect
on young women and negatively affect their
quality of life. The argument that preserving
or restoring physical appearance can have pro-
found psychological benefits provides the ba- .
sis for medicalizing cosmetic disorders such as
hair loss, hirsutism, or aging skin wrinkling
and discoloration.
5.1 Clinical Use
5.1.1 Current Drugs. Table 9.6 lists the
current therapies for hair growth disorders.
The effects of the two hair growth agents
(minoxidil and finasteride) have not been di-
rectly compared in clinical trials, and no clin-
ical studies have been performed on the com-
bination of the two drugs either, but a study in
an animal model for male pattern hair loss
suggests that the combination of 0.5 mglday
oral finasteride and 2% topical minoxidil has
an additive effect on hair growth (82, 83). Ef-
lornithine is the only drug currently approved
for the reduction of unwanted facial hair in
women. In addition to approved therapies,
434
hormonal treatments are still commonly used
both for female pattern androgenetic alopecia
and hirsutism. Estrogens (ethinylestradiol
or oral contraceptives) and antiandrogens
(cyproterone acetate, spironolactone, and flu-
tamide) are used either alone or in combina-
tions for either condition (73,81). Finasteride
was found to be effective for the treatment of
idiopathic hirsutism at a dose of 5 mglday, al-
though it is not approved for the indication.
This section will mainly focus on the two ap-
proved hair growth agents.
5.1.2 Adverse Effects. The side effects of
topical minoxidil are mainly local, caused by
skin irritation and contact dermatitis. Sys-
temic side effects are uncommon because of
limited percutaneous absorption, but diffuse
hypertrichosis of the face and limbs has been
reported with the 5% solution and was attrib-
uted to systemic absorption of the drug (84).
Although topical minoxidil does not change
blood pressure in healthy subjects, it increases
heart rate by 3-5 beatslmin and slightly in-
creases the left ventricular end-diastolic vol-
ume, cardiac output, and left ventricular mass
(85). These effects are not considered clini-
cally significant, and the potential for cardio-
vascular side effects is very low.
The clinical dose of finasteride is well toler-
ated. The main side effects reported in phase
I11 clinical studies were sexual function disor-
ders including decreased libido, ejaculation
disorders, and erectile dysfunction. All these
sexual disorders were mild-to-moderate and
were reversed on discontinuation of the drug,
and in some patients, they resolved even with
continued therapy (86).A 5-mgdose in hirsute
women was well tolerated with no significant
side effects, although the risk of developing
abnormalities in the external genitalia of male
fetus, if the drug is taken or a crushed tablet is
handled by a pregnant woman, causes a great
limitation to its use in women at child-bearing
age.
Topical eflornithine has a very good safety
profile. The only side effects observed in the
clinical trials at a higher frequency than pla-
cebo were related to skin irritation and in-
cluded stinging or burning skin and rash at
the site of application.
Lifestyle and Over-the-counter Drugs
5.1.3 Pharmacokinetics. Minoxidil is poorly
absorbed through skin, and systemic accumula-
tion after topical application is unlikely (87). Af-
ter topical application, minoxidil appears in the
systemic circulation at clinically insignificant
levels. The total amount recovered in urine is
less than 4% of the applied dose. Applying the
drug to the entire scalp is equivalent to a sys-
temic dose of 2.4-5.4 mglday (88). A total of 41-
45% of the applied dose remains on the scalp or
is lost on the pillowcase. Dermal metabolism of
minoxidil is negligible (89). Systemic minoxidil
elimination, after topical administration, is zero
order, which indicates that it is controlled by
zero order percutaneous absorption. The main
route of elimination after oral absorption is he-
patic metabolism. The primary metabolite is a
glucuronic acid conjugate at the N-oxide posi-
tion of the pyrimidine ring. Twenty percent of
the orally administered drug is excreted un-
changed in urine, and 95% of the total dose is
recovered in urine within 48 h.
Finasteride is rapidly absorbed after oral
administration, with peak plasma concentra-
tion reached in 1-2 h. The oral bioavailability
is about 80% and is not dose-dependent. Food
has no effect on the bioavailability of finas-
teride. Moderate accumulation occurs with
multiple dosing, and steady state is reached
within 3 days. Finasteride has a large voluhe
of distribution (76 L) as a result of wide tissue
distribution. Approximately 90% of the circu-
lating drug is bound to plasma proteins. Fin-
asteride undergoes extensive hepatic metabo-
lism. Finasteride metabolism occurs in the
liver through an oxidative pathway by cyto-
chrome P450 3A4 enzymes. The two major
metabolites are w-hydroxfinasteride and a
monocarboxylic acid derivative. Their in vitro
activity is less than 20% of that of finasteride,
but their i n vivo activity has not been studied.
Virtually no unchanged drug is excreted in
urine; 56.8% of the metabolized drug is ex-
creted in bile and 39.1% in urine. The mean
elimination half-life after multiple oral dosing
is 4.8 h, and the mean clearance after intrave-
nous infusion is 9.9 L/h. The mean serum half-
life is about 6 h in middle-aged men and
slightly longer in the elderly, but dosage ad-
justment is not necessary (86,90). The biolog-
ical half-life of finasteride is longer than its
5 Hair Growth Disorders
serum half-life. After discontinuation of the
drug, dihydrotestosterone (DHT) takes at
least 2 weeks to return to baseline levels.
Eflornithine absorption after percutaneous
administration is minimal (<I%of the applied
dose). Commonly used hair removal methods
such as shaving, plucking, or tweezing, when
performed within 2 h before application, did
not increase the percutaneous absorption.
Steady state is reached after 4 days of twice
daily application. Eflornithine is excreted un-
changed in urine, and no metabolism has been
observed. The apparent steady-state half-life
is approximately 8 h.
5.2 Physiology and Pharmacology
5.2.1 Physiology of Hair Growth. Hair
growth goes through a three-phase cycle (74):
anagen (growth phase), catagen (involution),
and telogen (rest). At the end of the telogen
phase, hair is shed and the next cycle begins. In
a normal scalp, approximately 90-95% of hair is
in the anagen phase. The duration of anagen
determines the hair length. Hair diameter is de-
termined by the volume of the hair bulb. The
scalp's terminal hair follicles are predetermined
to grow long thick hair, whereas the vellus hair
follicles on most of the body are predetermined
to grow short and h e hair. In androgenetic al-
opecia, the cycle of scalp hair growth is altered,
with gradual reduction in the length of anagen
phase leading to a reduction in the ratio of ana-
gen to telogen hair (86). This leads to progres-
sive miniaturization of the scalp hair in a recog-
nizable pattern. The main difference between
the male and female androgenetic alopecia,
other than the visible pattern, is that bald men
have reasonable hair densities (hairs per square
centimeter) even though the hairs in bald areas
are predominantly vellus (short and thin), with
some short hairs that have normal diameters. In
women with androgenetic alopecia, although
the scalp has fewer hairs per square centimeter,
the hair that remains is similar in diameter and
length to hair fibers in non-balding women (73).
It is not clear if these differences have any impli-
cations on the pharmacological therapy of the
condition.
5.2.2 Role of 5-a-Reductase. Skin is a tar-
get tissue for androgens; androgen receptors
are present in the hair follicles and sebaceous
glands of the scalp skin. Although testoster-
one acts directly on androgen receptors in sev-
eral tissues such as skeletal muscles, male
beard hair follicles, and male external genita-
lia, it has to be metabolized to the more potent
androgen dihydrotestosterone to act on andro-
gen receptors in the scalp hair follicles and
prostate among other tissues. 5a-reductase is
an important enzyme that mediates testoster-
one intracellular conversion to dihydrotestos-
terone (DHT). There are two types of 5a-re-
ductase. Type I is primarily present in the
skin, especially the sebaceous glands, epider-
mal and follicular keratinocytes, and sweat
glands, as well as in the liver and kidneys.
Type I1 is mainly found in the gonadal tissue
(prostate, seminal vesicles, and fetal genital
skin), dermal papilla, and in the liver and scalp
hair follicles (91, 92). Recent evidence proved
that type I1 exists in the root sheaths of the
scalp hair follicles and predominates in the
dermal papillae, contrary to the earlier studies
that suggested predominance of type I at this
location (86,92).5a-reductase converts testos-
terone to dihydrotestosterone, the androgen
responsible for androgenetic alopecia. Type I 5
a-reductase is responsible for one-third of cir-
culating DHT, whereas the type I1 isoenzyme ,
is responsible for two-thirds of circulating
DHT (93). The active androgen DHT binds
androgen receptors in the susceptible hair fol-
licles. The hormone-receptor complex binds to
specific gene sites to stimulate gradual trans-
formation of large terminal follicles to minia-
turized follicles (74, 91).
5.2.3 Mechanisms of Action. The two ap-
proved hair growth agents act by totally differ-
ent mechanisms. Minoxidil, the older agent,
acts by stimulating the microcirculation in the
balding scalp, whereas finasteride, the newer
agent, acts by altering androgen metabolism
in the hair follicles, through inhibition of type
I1 5a-reductase.
5.2.3.1 Minoxidil. Minoxidil increases the
blood flow to the follicular dermal papilla by a
direct vasodilation effect on the arteriolar
blood vessels (94, 95). Vasodilation is caused
by the active metabolite minoxidil sulfate.

Minoxidil is converted to its active metabolite
minoxidil sulfate by liver sulfotransferase sul-
fation (96). Minoxidil sulfate acts directly on
the cutaneous blood vessels and induces
smooth muscle relaxation. It has been sug-
gested that minoxidil sulfate acts as a potas-
sium channel agonist to enhance potassium
permeability. This results in hyperpolariza-
tion and causes reduction in agonist-stim-
ulated Ca2+ influx and hence decreased
cytoplasmic free Ca2+ concentration, and
therefore causes smooth muscle relaxation
(97, 98). Minoxidil sulfation occurs both in
proliferating keratocytes and in hair follicles,
which suggests that an additional mechanism
that results in direct stimulation of the hair
follicle independent of vasodilatation might be
involved (99, 100). In vitro data suggest that
the direct effect of minoxidil involves in-
creased biosynthesis of glycosaminoglycans
(100). It has also been shown that minoxidil
inhibits lysyl hydroxylase, an enzyme that cat-
alyzes the hydroxylation of certain lysine res-
idues in the polypeptide precursors of procol-
lagen (91). Hydroxylysine is important for the
formation of intermolecular crosslinks in the
collagen fiber. It is not known whether mi-
noxidil's effect on hair growth is related to its
effect on collagen metabolism.
Minoxidil increases the duration of anagen
hair and causes the growth of larger and nor-
mally formed follicles compared with the pre-
treatment short miniaturized terminal hair
follicles, which yields thicker longer hair and
decreased shedding. Minoxidil, however, does
not increase the total hair count (74, 95). Be-
cause of the non-specific mechanism of min-
oxidil, the drug can promote hair growth re-
gardless of the cause of hair loss. In addition to
its effectiveness in androgenetic alopecia, it
promotes hair growth in unrelated conditions
such as alopecia areata, congenital hypotri-
chosis, and loose androgen syndrome (74).
Vertex hair loss responds better to minoxidil
therapy than frontal hair loss. The effect of
minoxidil seems to plateau after approxi-
mately 1 year of therapy and reverses when
the treatment is stopped.
5.2.3.2 Finasteride. Finasteride is a spe-
cific and competitive inhibitor of type I1 5a-
reductase. It is 100-fold more selective to the
Lifestyle and Over-the-counter Drugs
type I1 isoenzyme compared with type I (101).
It has been shown through in vitro studies
that finasteride competes with testosterone
for the same binding site on 5a-reductase, but
it has no effect on the binding of testosterone
or DHT on androgen receptors (102). The in-
hibition of type I1 5a-reductase blocks the pe-
ripheral conversion of testosterone into DHT
and therefore results in decreased DHT con-
centration both in the circulation and in tar-
get tissue. Sixty-five percent suppression of se-
rum DHT is reached within 24 h of oral dosing
with a 1-mgtablet (93),and a median of 68.4%
reduction occurred in men who were treated
for 1 year (86). Therapeutic doses of 0.2-5 mgl
day for 4-6 weeks reduced the scalp DHT
level by up to 65%. The relative contributions
of the reduction in scalp DHT and tissue DHT
to finasteride effect on male pattern baldness
has not been identified. The circulating levels
of testosterone and estradiol increase slightly
but remain in the normal range. Finasteride
does not have any direct androgenic or antian-
drogenic effect and does not cause any signifi-
cant changes in the levels of luteinizing hor-
mone (LH)and follicle stimulating hormone
(FSH). The therapeutic dose of finasteride (1
mglday) causes significant increases in hair
count and hair growth both in frontal hair loss
and vertex hair loss male subjects (86). As &h
minoxidil, finasteride does not revive hair fol-
licles that are inactive (i.e., in older men who
are bald) and is therefore recommended to be
used only by younger men with partial hair
loss. At least 3-4 months of therapy with ei-
ther minoxidil or finasteride is needed before
any hair growth effect can be seen. The ther-
apeutic effect declines when treatment is dis-
continued, with complete reversal within a
few months in the case of minoxidil and 12
months in the case of finasteride (86,93).
5.2.3.3 Eflornifhine. Eflornithine acts by
inhibiting the enzyme ornithine decarboxyl-
ase (ODC) in the hair follicles of the human
skin (93).The enzyme is necessary for the syn-
thesis of polyamines. Animal data indicates
that inhibiting ODC inhibits cell division and
synthetic function and therefore inhibits hair
growth. It is postulated that eflornithine
causes irreversible inhibition of the enzyme.
5 Hair Growth Disorders

5.3 Chemistry and Structure-Activity 5.3.2 5cu-Reductase Inhibitors. Finasteride

Relationships (2) is a Cazasteroid derivative of the 3-0x0-5a-

5.3.1 Minoxidil. Minoxidil (1)is 2,4-dim-

ino-6-piperidino-pyrimidine-3-oxide. It was
developed as an antihypertensive from triam-
inotriazines. Triaminotriazines were found to
be potent vasodilators in dogs and cats because

(2) Finasteride

steroids. An important structure requirement

(1) Minoxidil
of the formation of N-oxides. In humans, how-
ever, they were inactive because of the inability
to form N-oxides. This led to the development of
minoxidil by isosteric replacement by triamin-
opyrimidine of a triaminotriazine moiety.
The structural requirements for minoxi-
dil's hypotensive effect (103), and its lysyl hy-
droxylase inhibitory effect (91) have been re-
ported, but the relevance of these requirements
to the hair growth effect of minoxidil is un-
known. One of the distinct features of minoxi-
dil is the nitroxide group in position 1 of the
pyrimidine ring. This group is essential for the
vasodilator effect. However, for the effect on
lysyl hydroxylase, removal of the nitroxide ox-
ygen results only in partial loss of activity. A
secondary or tertiary m i n e substituent on po-
sition 4 of the pyrimidine ring is also required
for hypotensive activity. In minoxidil, this re-
quirement is met by a piperidinyl group. Re-
placing the piperidinyl group with another
source for a tertiary or secondary nitrogen
such as pyrrolidinyl, morpholinyl, or an
N-methylpiperazinyl group does not cause
detrimental effects on the antihypertensive
activity but results in total loss of lysyl hydrox-
ylase inhibitory activity. Hydroxylation at po-
sition 3 or 4 of the piperidine ring (by meta-
bolic transformation) results in marked
reduction in hypotensive activity. The same 3-
or 4-hydroxy piperidinyl derivatives are as po-
tent as the parent compound in suppressing
lysyl hydroxylase.
for 5a-reductase inhibition of azasteroids is
stable configuration in ring A of the steroid
molecule that mimics the transition state in
the conversion of testosterone to DHT (104,
105). This allows for a tight binding between
the inhibitor and the active site of the enzyme.
Most of the 5a-reductase inhibitors in devel-
opment are either 4- or 6-azasteroids. How-
ever, steroidal carboxylic acids and non-steroi-
dal tricyclic compounds with structural
resemblance to azasteroids are also potent in-
hibitors of 5a-reductase (106).Molecular mod-
eling of the binding between the enzyme and
substrates suggests that there is a require-
ment for groups to mimic the carbonyl group
of ring-A in the steroid structure (107). An
interesting nonsteroidal series of compounds
that satisfy this requirement is benzoquinoli-
nones. Among this class is Lilly's LY 191704
(3),a potent non-competitive 5a-reductase in-
hibitor with high selectivity to type I
isoenzyme.
The 4- or 6-aza moiety is another important
structural requirement. A notable exception
438
to this requirement is epristeride (4), a type I1
5a-reductase inhibitor with no nitrogen in the
steroidal ring structure. The position of this
d.
0
HOOC
(4) Episteride
nitrogen in the steroidal skeleton does not af-
fect the potency of 5a-reductase inhibition or
isoenzyme selectivity. Most of the 4- and
6-azasteroids that have been reported have
higher selectivity to isoenzyme 11, with only
few exceptions (e.g., Merck's L 733692). The
position of the aza moiety seems to affect the
mechanism of inhibition (108). It has been re-
ported that the inhibition of 5a-reductase by
6-azasteroids is reversible, whereas the 4-aza-
steroid derivatives such as finasteride cause
irreversible inhibition. Benzoquinolinones have
a nitrogen that mimics that of 6azasteroids.
In Cazasteroids, a methyl group on the nitro-
gen produces greater activity than a hydrogen.
A A1 double-bond at the C1-C2 position (in
ring A) results in substantial loss of inhibitory
action on hair follicle 5a-reductase. However a
A5 double-bond in ring D has little effect on
enzyme inhibition.
A bulky lipophilic moiety on C-17 is an-
other structural requirement for maximum
5a-reductase inhibition. A long C-17P side-
chain increases the activity (101). Replacing
the tert-butyl group on the C17 amide nitrogen
of finasteride with a phenyl group results in a
slight decrease in potency against type I1
isoenzyme and an increase in the potency
against type I (109). Further introduction of
an alkyl group on the same nitrogen results in
a drastic loss of activity against both isoen-
zymes because of changes in the conforrna-
tional preference (from trans to cis). 6-Azaste-
roids with C17 anilide substitution are also
potent inhibitor of the enzyme. 2,5-Disubsti-
tution of the phenyl ring in the anilide series
Lifestyle and Over-the-counter Drugs
increases the potency against type I 5a-reduc-
tase while retaining higher potency against
type I1 (110). An arylcycloalkyl group on the
C17 amide of 6-azasteroids similarly results in
high potency against both isoenzymes with
higher selectivity to type 11.
The interest in pharmacological therapy for
male pattern hair loss started as early as 1965,
when a topical preparation of testosterone was
tested for presumed benefits. Topical testos-
terone failed to show any efficacy (86). The
interest then declined until reports about
stimulation of hair growth in hypertensive pa-
tients being treated with the then experimen-
tal drug minoxidil renewed the hopes.
The initial research leading to the discov-
erv
" of minoxidil started in 1960 when re-
searchers from the Upjohn Company per-
formed empirical pharmacological screening
on N,N-diallylmelanine (DAM)-a compound
they ordered from the American Cyanamid
chemical catalog (111).The first pharmacolog-
ical action that was observed was reduction of
gastric acidity in pyloris-ligated rats, but after
oral administration to dogs, DAM produced a
delayed and very prolonged blood pressure re-
duction. However, a subsequent clinical trial
showed no clinical effect in hypertensive pa-
tients. This led to the identification of amac-
tive metabolite in dogs that was absent in hu-
mans and was responsible for the hypotensive
effect of DAM. Subsequently, this metabolite
(DAMN-0) was synthesized, and it was deter-
mined that it acted by direct vasodilatation. A
single-dose clinical trial that was conducted in
late 1961 showed a marked reduction in blood
pressure and a long duration of action that
exceeded 24 h. Consequently, a multiple dose
study was initiated. A combination of salt re-
tention side effect and a mistake in interpret-
ing instructions led to the discontinuation of
the study. The situation was further compli-
cated by the discovery of a cardiac lesion that
developed in a long-term toxicology study in
dogs. All this led to the loss of drug candidacy
of DAMN-0. Despite this, Upjohn researchers
continued to be interested in the direct vaso-
dilator action of DAMN-0 and continued their
search for better drug candidates. During the
following years, they synthesized hundreds of
5 Hair Growth Disorders
analogs, all bearing the N-oxide moiety. Ten
candidates were selected and tested in dogs. It
was found that the same side effects were as-
sociated with the entire class of compounds
(salt and water retention, tachycardia, and
cardiac lesion). Upjohn researchers selected
minoxidil as the best candidate and decided to
proceed with clinical trials cautiously and only
in patients with life-threatening drug-refrac-
tory hypertension. The Investigational New
Drug Application of minoxidil was filed in
1968. The initial clinical trial showed dra-
matic reduction in blood pressure of severely
hypertensive and drug-refractory patients.
The expected side effects (salt retention and
tachycardia) occurred, but were reversed by
co-administration of diuretics and P-blockers,
respectively. The following trial demonstrated
superiority over hydralazine and led to wide-
spread investigators' interest in minoxidil. As
a result, the FDA approved an emergency-use
protocol in late 1970. Initially, each case had to
be approved by an FDA clinical reviewer, but
by 1971, an Upjohn clinical monitor was au-
thorized to approve the treatment. Gottlieb et
al. (112),the clinical investigators of these ini-
tial clinical trials, was the first to report the
hair growth stimulating effect of minoxidil.
Two dermatologists who were consulted about
the phenomenon decided to test a topical solu-
tion on the upper arm and were able to dem-
onstrate localized action at the site of applica-
tion (111). In 1971, Upjohn started absorption
and toxicology testing of the minoxidil topical
solution. In 1972, the first case of hair growth
on a bald scalp of a severely hypertensive pa-
tient who was being treated with minoxidil
was reported (111).Because of the continuing
concerns over oral minoxidil's side effects, Up-
john delayed the filing of topical minoxidil un-
til it gathered convincing evidence of safety
that was sufficient for approval of oral minoxi-
dil. The IND for the topical solution was filed
in 1977, and the FDA approval of oral minoxi-
dil for hypertension came in 1979. This recog-
nition of safety made it possible for Upjohn to
continue pursuing the hair growth indication.
Initial hair growth clinical trials began in
1978. Although these studies provided some
evidence of clinical efficacy, with hair growth
stimulation in a large percentage of subjects,
statistically significant differences were not
demonstrated until the project moved to large
phase 111multi-center clinical trials (111). Up-
john's efforts were culminated in 1988 by the
approval of minoxidil 2% topical solution for
male pattern androgenetic alopecia. In 1991,
the FDA approved it for female pattern hair
loss. The OTC switch of minoxidil topical so-
lution was approved in 1996 after an NDA ap-
plication. In 1997, the FDA granted its ap-
proval of 5% minoxidil topical solution for
initial marketing as an OTC medication. Min-
oxidil(5%) was never marketed as a prescrip-
tion drug.
Unlike minoxidil, the discovery of finas-
teride was a typical case of modern drug dis-
covery-a drug designed to target specific re-
ceptors that mediate the disease it is aimed at.
The first step that led to the development of
finasteride was discovery of the role of 5a-re-
ductase, which provided a better understand-
ing of the separate roles of testosterone and
DHT. The role of 5a-reductase was under-
stood through studies on the genetic disorder
5a-reductase deficiency, which produces a
form of male pseudohermaphroditism (106,
113, 114). Those studies suggested that the
clinical signs of the disorder imply that 5a-
reductase has a role in conditions such as
benign prostatic hyperplasia, androgenetic al-
opecia, acne, and hirsutism (106). Conse-
quently, researchers at Merck synthesized a .
series of azasteroids to target the enzyme and
hence provide potential treatments for these
conditions. After the isolation of the two 5a-
reductase isoenzymes, it was found that type
I1 5a-reductase, which is inhibited by finas-
teride, is the isoenzyme responsible for most
of the effects ascribed to 5a-reductase. Fur-
thermore, it was found that the patients with
male pseudohermaphroditism caused by 5a-
reductase deficiency are only deficient in type
I1 and have normal levels of type I isoenzyme
(114). Finasteride was first administered to
humans in early 1986. Biochemical efficacy
was demonstrated in the first clinical study
with marked reduction in circulating DHT
levels (102). Clinical studies were conducted
on finasteride for benign prostate hyperplasia
(BPH), hirsutism, male-pattern baldness, and
acne. The drug was first marketed for benign
prostatic hyperplasia as a 5-mg tablet under
the brand name Proscar after its FDA ap-

proval in 1992. The hair growth indication
was being pursued at the same time. Propecia,
a 1-mgfinasteride tablet was approved for the
treatment of male-pattern hair loss in 1997.
Eflornithine (5) was originally developed
by Merrell Dow (now Aventis) as an antineo-
(5) Eflornithine Hydrochloride
plastic and antiprotozoal agent. It was ap-
proved in 1990 in the intravenous form for the
treatment of sleeping sickness (trypanosomi-
asis) under the brand name Ornidyl. Inhibi-
tion of hair growth was observed during the
clinical trials. The indication was licensed to
Bristol-MyersSquibb (BMS),which partnered
with Gillette on the development program for
reduction of unwanted hair in women. Gillette
did the early development work and BMS con-
ducted the clinical trials and handled regula-
tory filing. Eflornithine gained FDA approval
in July 2000 and was subsequently launched
in September of the same year. An OTC switch
of eflornithine is a very likely long-term goal.
5.5 Current and Future Trends
Both androgenetic alopecia and hirsutism are
very active areas of research. With the in-
creased knowledge of the biochemical and
physiological basis of the conditions, novel
compounds are being designed to target spe-
cific enzymes or receptors that are involved.
Most of the compounds under development
fall into three major categories: 5 a-reductase
inhibitors, topical antiandrogens, and potas-
sium channel agonists. The first two mecha-
nisms are targeted for both scalp hair growth
stimulation and bodylfacial hair growth inhi-
bition. The third category is specific for hair
growth promoting agents. Potassium channel
agonists are believed to induce local vasodila-
tation and therefore enhance the microcircu-
lation in the scalp similar to minoxidil sulfate.
They cause smooth muscle relaxation by hy-
perpolarizing the cell membrane and inhibit-
ing acetylcholine-activated voltage-sensitive
Lifestyle and Over-the-counter Drugs 1
Ca2+influxes and thus attenuating the acetyl-
choline-induced tonic contraction.
Among the most promising 5 a-reductase
inhibitors is Glaxo's dutasteride (GG-745, GI-
198745). Glaxo SmithKline has submitted an
NDA with the FDA for BPH in 2001. Dutast-
eride, a 4-azasteroid, is an inhibitor of both
type I and type I1 isoenzymes and is in phase I1
clinical trials for androgenetic alopecia. Table
9.7 lists some of the compounds in various
stages of development for hair growth disor-
ders. Many of these compounds, particularly
antiandrogens and 5 a-reductase inhibitors in
earlier phases of development, are being
tested for multiple indications.
6 SEXUAL DISORDERS
The recent extraordinary interest in sexual
disorders was primarily spurred by the intro-
duction of sildenafil (Viagra), the first effective
oral medication for erectile dysfunction. The
unparalleled global media hype that followed
the launch of Viagra greatly increased the
public awareness of erectile dysfunction and of
sexual disorders in general. The enormous
public response to sildenafil also stirred a de-
bate over the reimbursement of such thera-
pies and of lifestyle drugs in general. Several
health authorities argued that impotence i s
not a disease and does not pose any health risk
to the individual or the society. To counter this
argument, drug companies that are involved
1
in the field of sexual disorders intensified their
efforts in gathering evidence on the impact of
sexual dysfunction and its successful treat-
ment on the quality of life of the individuals
and their partners. This gave a new life to
other sexual disorders that, until recently,
were overlooked by the public and researchers
as well. The most common sexual disorders
are erectile dysfunction, premature ejacula-
tion, and female sexual dysfunction.
Erectile dysfunction, also known as impo-
tence, is defined as the inability to achieve or
maintain an erection adequate for sexual sat-
isfaction. It is estimated that 30 million men in
the United States have partial or total erectile
dysfunction (117). The number is expected to
increase as the American population contin-
ues to age. Studies have shown that over 50%
6 Sexual Disorders

Table 9.7 Compounds in Various Stages of Development for Hair Growth

Disorders (115,116)
Generic Name/
Laboratory Code Originator Chemical Class Mechanism of Action Indication
P-1075 Leo (Denmark) F'yridyl Potassium channel Alopecia
cyanoguanidine agonist
Dutasteride Glaxo-Smithkline 4-Azasteroid 5 a-Reductase Alopecia, BPH
inhibitor
Tricomin ProCyte Tripeptide copper Unknown Alopecia
(PC 1358) compounds
Turosteride Pharmacia Non-steroidal BPH, alopecia,
teteracyclic acne,
structure closely hirsutism
resembles 4-
azasteroid
CS 891 Sankyo (Japan) Unknown 5 a-Reductase BPH, alopecia
inhibitor
L 733692 Merck Type I 5 a-Reductase Male pattern
inhibitor baldness
MK 434 Merck 5 a-Reductase BPH, alopecia,
inhibitor acne
EM 250 Endorecherche Antiandrogen Acne, alopecia
(SCH 54726) (Canada) and
Schering
Plough
Aventis Benzonitrile Non-steroidal topical Acne, alopecia
derivative antiandrogen
Lilly Benzoquinolinone Type 1 5 a-reductase BPH, alopecia,
inhibitor acne
Chugai (Japan) Carbothiomide Potassium channel Alopecia
agonist
Chugai Unknown Potassium channel Alopecia .
agonist
Ligand Unknown Non-steroidal Alopecia,
antiandrogen hirsutism,
acne, BPH

of men ages 40-70 have some degree of erec-
tile dysfunction, with 15%suffering from com-
plete erectile dysfunction (118).
Premature ejaculation is another wide-
spread male sexual disorder. It is defined as
the persistent or recurrent ejaculation of se-
men with minimal sexual stimulation before,
on, or shortly after vaginal penetration, and
before the person or his partner desires it
(119). Although less publicized than erectile
dysfunction, premature ejaculation is believed
to be the most common male sexual disorder.
The prevalence rate.is estimated at 30-40% of
"normal" men (120).
Female sexual dysfunction encompasses
a wide range of little understood disorders.
These include the following: hypoactive sex-
ual desire disorder. sexual arousal disorder,
orgasmic disorder, and sexual pain disor-
ders. The overall prevalence of female sexual
disorders is estimated a t 30-50% (121, 122).
The U.S. National Health and Social Life
Survey of women ages 18-59 reported a
prevalence rate of 43% (123). Until recently,
most of the female sexual disorders were
considered part of the natural aging process,
and the subject gained very little attention
from researchers and the public. The only
available treatment was counseling. This
has changed recently, as a result of the soar-
ing interest in sexual disorders and lifestyle
drugs in general, that was fueled by the phe-
nomenal success of Viagra. It is now widely
recognized that female sexual dysfunction is
442
Table 9.8 Currently Approved Drugs for Erectile Dysfunction
Generic Name Brand Name Originator
Sildenafil citrate Viagra Pfizer
Alprostadil Muse Vivus
Alprostadil Caverject Upjohn
Yohimbine Aphrodyne N/Aa
"Yhimbine and Yohimbe were used in folk medicine as aphrodisiacs for a long time.
an important health issue that affects the
quality of life of millions of women.
6.1 Clinical Use
6.1.1 Current Drugs. Several pharmaco-
logical agents are available for the treatment
of erectile dysfunction with different etiolo-
gies, which makes the condition treatable in
the vast majority of sufferers. Table 9.8 lists
the pharmacotherapeutic agents currently ap-
proved for erectile dysfunction.
The guidelines for management of erectile
dysfunction issued by the First International
Consultation on Erectile Dysfunction recom-
mends involving the patient in selecting the
treatment of his preference after explaining
the benefits, risks, and costs involved with
each treatment option (124). The non-inva-
siveness and convenience of oral medication
makes it the first choice by the vast majority of
patients unless contraindicated. Sildendl is
currently the most prescribed treatment for
erectile dysfunction. It is effective as a single
dose and is recommended to be taken approx-
imately 1 h before sexual activity, but can be
taken anywhere from 30 min to 4 h before
sexual activity. The overall efficacy of silde-
nafil, defined as percentage of successful at-
tempts at sexual intercourse, is approximately
70% (125).
The leading intracavernosal agent is al-
-
wostadil. It is indicated for the treatment of
erectile dysfunction caused by neurogenic,
vasculogenic, psychogenic, or mixed etiology.
Erection occurs in 5-10 min after intracaver-
nosal injection of alprostadil. In clinical prac-
tice, the dose is individualized and titrated
slowly to the lowest possible effective dose to
avoid prolonged erection and priapism (erec-
tion that lasts for more than 6 h), which can be
potentially serious side effects. The target is
an erection that lasts for approximately 1 h.
Lifestyle and Over-the-counter Drugs
Dosage Form Dosage Strength
Oral tablet 25,50,100 mg
Urethral suppository 125,250,500,1000 pg
Intracavernosal injection 5,10,20,40 pg
Oral tablet 5.4 mg 3 times a day
Intracavernosal alprostadil is still the most ef-
fective treatment, although its use is limited
by the side effects and the inconvenience of
self-injection and rapid onset of action, which
results in an unnatural erection. More than
90% of alprostadil intracavernosal injections
result in successful sexual intercourse (126).
Transurethral alprostadil is a micro-supposi-
tory that is inserted into the stem of the ure-
thra using an applicator. Although it is a more
convenient route of administration, its overall
efficacy is about 50% (126, 127).
The vasoactive amines phentolamine and
papaverine are occasionally used as intracav-
ernosal therapy, usually in combination with
alprostadil, although their use for erectile dys-
function is off-label. Moxisylyte is another va-
soactive agent used as intracavernosal ther-
apy. The drug is approved in several European
countries, but is not approved in the United
States. The advantages over alprostadil are
that with moxisylyte, sexual stimulation is
still required to achieve full erection and that
detumescence occurs on ejaculation.
Yohimbine is a moderately effective and
well-tolerated oral agent for erectile dysfunc-
tion, yet it has not been adequately evaluated
in well-designed placebo-controlled studies,
although two meta-analyses of the few ran-
domized placebo-controlled studies demon-
strated its advantage over placebo (128-130).
It is more effective in patients with erectile
dysfunction of psychological etiology (131,
132). Yohimbine is a registered drug in the
United States. The label indications are sym-
patholytic and mydriatic, but the label also
states "impotence has been successfully
treated with yohimbine in male patients with
vascular or diabetic origins and psychogenic
origins (18mglday)" (93). One of the disadvan-
tages of yohimbine is its daily dosing schedule,
which in addition to being inconvenient, may
6 Sexual Disorders
contribute to the low efficacy observed in some
of the clinical studies. A single on-demand
dose has not been evaluated, although it has
been successfully used by some clinicians and
seems to be at least as effective as the standard
daily dose (128, 132). Moreover, it has been
suggested that tolerance develops after
chronic administration of yohimbine, which
further diminishes the efficacy of a daily dose
(128). Because of the lack of conclusive clinical
data on yohimbine, the American Urological
Association, in its guidelines on the treatment
of organic erectile dysfunction issued in 1996,
concluded that "based on the data to date, yo-
himbine does not appear to be effective for or-
ganic erectile dysfunction, and thus should
not be recommended as treatment for the
standard patient" (133).
Topical anesthetics are the only approved
treatment for premature ejaculation. Few un-
controlled clinical studies reported positive re-
sults (119, 134). However, the decades-old
therapy has not been evaluated in controlled
clinical trials. Oral pharmacotherapy has been
used off-label and is gaining more popularity
with the increased public interest in oral ther-
apies for sexual disorders. Some of the drugs
that have been historically used are adrener-
gic antagonists and y-amino butyric acid
(GABA). Selective serotonin reuptake inhibi-
tors, namely paroxetine and fluoxetine, are
currently the most commonly used oral agents
for premature ejaculation.
There are no approved treatments for fe-
male sexual dysfunction, although the subject
is currently enjoying vast interest. At present,
the main off-label treatments are the vasoac-
tive agents developed for erectile dysfunction.
Clinical studies are underway to support their
use.
The following sections will focus on ap-
proved treatments for erectile dysfunction.
6.1.2 Adverse Effects. Sildenafil is well tol-
erated in patients with normal cardiovascular
function. Most of the side effects reported in
the clinical trials at a higher rate than placebo
are mild and are related to the drug's vasodi-
latory effect. They include headache, flushing,
and nasal congestion. Other frequent side ef-
fects include abnormal vision (impairment of
color discrimination) and dyspepsia. The
former is a result of PDE6 inhibition and the
latter is caused by the inhibition of PDE5 in
the lower esophageal sphincter. Prolonged
erection has been reported but is less frequent
than with intracavernosal therapy. Priapism
was reported in few patients, but the risk rate
is extremely low. Potentially serious cardio-
vascular effects including decreased blood
pressure caused by systemic vasodilatation
and decreased cardiac output may occur in pa-
tients with preexisting cardiovascular risk fac-
tors. Sexual activity is an added risk factor in
those patients. In addition, sildendl potenti-
ates the hypotensive effects of nitrates and is
contraindicated in patients who are using or-
ganic nitrates. Most of reported deaths with
sildendl were caused by the concomitant
treatment with nitrates. Some reported
deaths occurred in patients with preexisting
cardiovascular risk during or after sexual
activity.
The main side effects of alprostadil are lo-
cal. For intracavernosal injection, these in-
clude the following: penile pain after injection,
prolonged erection, priapism, painful erec-
tion, penile fibrosis, and injection site hema-
toma. Priapism and prolonged erection are
more serious side effects with alprostadil than
with sildendl. The local side effects after in-
traurethral administration are usually milder .
and include penile pain, urethral discomfort,
and urethral bleeding/spotting and prolonged
erection. Systemic side effects include hypo-
tension (intraurethral administration only),
headache, dizziness, and upper respiratory
tract infections.
Yohimbine is well tolerated at the oral
doses used for erectile dysfunction. The main
side effects are nausea, dizziness, and ner-
vousness. Headache and skin flushing have
also been reported. Yohimbine has no signifi-
cant effect on P-adrenergic receptors, and its
effect on blood pressure has not been ade-
quately evaluated. Common side effects after
parented administration include sweating,
nausea, and vomiting. Yohimbine penetrates
the blood-brain barrier and can produce a
complex pattern of responses (93). The central
effects include anti-diuresis, a general picture
of central excitation including elevated blood
pressure and heart rate, increased motor ac-
tivity, irritability, and tremor.

6.1.3 Pharmacokinetics
6.1.3.1 Sildenafil. Sildenafil is rapidly ab-
sorbed after oral administration. The peak
plasma concentration is reached after 30-120
min of dosing, but the absorption rate is de-
layed if taken after high fat meals. The abso-
lute oral bioavailability is approximately 40%.
The mean steady-state volume of distribution
is 105 L, which indicates distribution into tis-
sues. Sildenafil and its major metabolite are
approximately 96% bound to plasma proteins.
Sildenafil is eliminated mainly by hepatic
metabolism. The main metabolizing enzyme is
cytochrome P450 3A4. The major circulating
metabolite is N-desmethyl sildenafil, which is
further metabolized. N-desmethyl sildenafil
retains sildenafil's selectivity profile and ap-
proximately 50% of its activity. Both sildenafil
and its major metabolite have terminal half-
lives of 4 h. The elimination half-life of silde-
nafil is 3-5 h. Most of the administered dose is
excreted in the feces as metabolites with only
13% excreted in the urine.
6.1.3.2 Alprostadil. Intraurethral PGE, (al-
prostadil) is absorbed from the urethra and
distributes to the erectile tissue (cavernosal
smooth muscles) by communicating blood ves-
sels between the corpus spongiosum and the
corpora cavernosa. The transurethral absorp-
tion is biphasic with an initial rapid phase fol-
lowed by slower absorption. Approximately
80% of the administered dose is absorbed
within 10 min and about 20% of the total dose
reaches the cavernosal tissue.
Alprostadil binds to plasma proteins, pri-
marily albumin and to a lesser extent to
a-globulin IV-4 fraction. There is no evidence
of tissue binding or accumulation.
Alprostadil is rapidly metabolized locally
within the corpus cavernosum and in the ure-
thra by enzymatic oxidation of the 15-hy-
droxyl group to 15-keto-PGE,. 15-Keto-PGE,
has only 1-2% of the biological activity of
PGE,. 15-Keto-PGE, is rapidly reduced to
the inactive metabolite 13,14-dihydro,15-
keto-PGE,, which is the most abundant me-
tabolite in plasma. DHK-PGE, is further me-
tabolized to other inactive metabolites. The
half-life of alprostadil after intracavernosal
administration is 5-10 min (135).Only a small
fraction of the dose reaches the systemic cir-
culation after intracavernosal or intraure-
Lifestyle and Over-the-counter Drugs
thral administration. The systemic metabo-
lism of alprostadil is also very rapid. Eighty
percent of the circulating alprostadil is metab-
olized in one pass through the lungs; thus,
plasma levels are low and become undetect-
able within 1 h of intraurethral or intracaver-
nosal administration. The main pathways for
systemic metabolism are p- and *oxidation.
The metabolites are excreted primarily by the
kidneys. Ninety percent of the intravenously
administered alprostadil appears in urine as
metabolites within 24 h after administration.
The remaining 10% is excreted in the feces.
6.1.3.3 Yohimbine. Yohimbine absorption
after oral administration is highly variable.
Both rapid and slow absorption have been re-
ported (136,137). Peak plasma levels occurred
at 10-45 min post-dosing in one study and
36-120 min with an average of 1.1 h in an-
other study. The oral bioavailability is gener-
ally low and highly variable. It ranges from 4%
to 87%. Yohimbine is highly metabolized, with
only 1% of the administered dose excreted un-
changed in urine. The mean elimination half-
life is 0.63-1.5 h. Clearance is highly variable
as well, with a mean value of 11 mL/min/kg.
Yohimbine has a steady-state volume of distri-
bution of approximately 32 L, and is highly
bound to plasma proteins (around 80%).It has
been postulated that an active metabolite with
a longer duration of action is at least partially
responsible for yohimbine pharmacological ac-
tivity (138, 139). Two metabolites have been
identified: 10-hydroxy-yohimbine and ll-hy-
droxy-yohimbine. Both the parent drug and
the two metabolites are distributed to the ce-
rebrospinal fluid. ll-Hydroxy-yohimbine was
shown to have a2-adrenoreceptor antagonist
activity in vitro. It has a long half-life of ap-
proximately 9.6 h and is present at a much
higher plasma concentration than yohimbine
after oral administration of the parent drug
(137).
6.2 Physiology and Pharmacology
6.2.1 Physiology of Erection. The penis is
composed of three bodies: a pair of corpora
cavernosa on the dorsal side and a corpus
spongiosum on the ventral side. The role of the
corpus spongiosum is to protect and support
the urethra, while the two corpora cavernosa
6 Sexual Disorders
are the parts that provide structure to the pe-
nis in the erect state. The cavernosal bodies
consist of a network of vascular sinuses sup-
plied by the terminal branches of the caver-
nosal arteries. The vascular sinuses are sup-
ported by smooth muscles that are normally
contracted when the penis is in the flaccid
state. Factors that mediate cavernosal smooth
muscle contraction, and therefore promote pe-
nile flaccidity, include the following (140):
a-adrenoreceptors, endothelin, angiotensin,
and thromboxane A,.
Erection occurs as a result of increased
pressure in the corpora cavernosa, which
translates into penile rigidity. The pressure
increase is caused by three synergistic pro-
cesses: (1)relaxation of the smooth muscles of
the corpora cavernosa; (2)increase in arterial
blood flow to the penis; and (3) restriction of
the venous blood flow out of the penis. Both
central and peripheral mechanisms contrib-
ute to the process of penile erection. At the
central level, the psychological component of
penile erection is controlled by the hypotha-
lamic and limbic systems (140). At the periph-
eral level, both sympathetic and parasympa-
thetic pathways as well as several mediators
are involved. Psychogenic and local stimula-
tion results in the release of neurotransmit-
ters from the cavernosal nerve terminals and
smooth muscle endothelium. Factors that me-
diate the corpus cavernosum relaxation in-
clude nitric oxide (NO), vasoactive intestinal
peptide (VIP), calcitonin gene-related peptide
(CGRP), and prostaglandin.
6.2.2 Role of Nitric Oxide. The N O path-
way is the best understood and is believed to
be the most important pathway for penile
erection. NO is released from nonadrenergic
noncholinergic (NANC) nerves in the corpus
cavernosum and from the endothelium that
lines the cavernosal vascular sinuses and
blood vessels (140). Nitric oxide synthase
catalyses NO formation from the precursors
L-arginine and molecular oxygen (141). NO
production in the vascular endothelium is
stimulated by muscarinic acetylcholinergic re-
ceptors. The released NO diffuses into smooth
muscles and interacts with guanylate cyclase,
which catalyses the formation of cyclic
guanosine monophosphate (cGMP) from
guanosine triphosphate. The resulting in-
crease in cGMP activates several processes
that lead to smooth muscle relaxation. The in-
creased arterial inflow to the corpora caver-
nosa as a result of smooth muscle relaxation
leads to an increase in intracavernosal pres-
sure and volume and thus increased penis
length and rigidity. The increase in intercav-
ernosal pressure results in compression of the
subtunical venules and hence reduction of the
venous outflow, which further increases pe-
nile rigidity (140, 142). cGMP activity is ter-
minated by hydrolysis by cGMP specific type-5
phosphodiesterase (PDE).
6.2.3 Other Mediators. It is believed that
VIP-, CGRP-, and prostaglandin-mediated
pathways contribute to smooth muscle relax-
ation by increasing intracellular CAMP con-
centration in the corpora cavernosa (140). In-
creased CAMP results in phosphorylation and
dephosphorylation of the actin-smooth-mus-
cle-myosin cascade that causes smooth muscle
relaxation. Prostaglandin E (PGE) causes
the elevation of CAMP through a G-protein-
coupled mechanism and activation of adenylyl
cyclase. In addition, PGE reduces the adreno-
receptor mediated vasoconstriction by inhibit-
ing norepinephrine release through prejunc-
tional receptors on norepinephrine containing
.
neurons.
6.2.4 Causes of Erectile Dysfunction. Erec-
tile dysfunction can result from neurological,
vascular, hormonal, or psychological factors,
or from a combination of two or more of these
factors. Neurological causes include spinal
cord injury, multiple sclerosis, or any other
condition that impedes the transmission of the
neural signal generated by psychogenic stim-
ulation. Vascular causes include arterial in-
sufficiency, which results in low arterial pres-
sure delivered to the penis, and venous
insufficiency, in which excessive venous out-
flow occurs because of inadequate compres-
sion of the subtunical venules. Erectile dys-
function of hormonal origin results from
inadequate androgenic stimulation of the sex-
ual center in the anterior hypothalamus,
which lowers libido and hence erection qual-
ity. In psychogenic erectile dysfunction, sexu-
ally inhibiting psychological issues can impede

stimulatory signals to the penis, and anxiety
can cause a sympathetic discharge, which fa-
vors flaccidity (142). It is usually difficult to
distinguish primary psychogenic erectile dys-
function because many men with organic erec-
tile dysfunction have a psychological response
to their condition.
6.2.5 Mechanisms of Action. Most erectile
dysfunction pharmacotherapies act by either
inhibiting the contractile system (e.g., a-adre-
noreceptor antagonists), or by stimulating or
enhancing the vasodilatory system (e.g., pros-
taglandin El, NO donors, PDE inhibitors).
These mechanisms can work regardless of the
origin of the disorder.
6.2.5.1 Sildenafil. Sildenafil is a selective
PDE5 inhibitor. PDE5 is the cGMP-specific
PDE and is the predominant form of the isoen-
zyme in the corpora cavernosa. By inhibiting
the hydrolysis of cGMP that is generated
through the NO pathway, sildenafil enhances
the cavernosal smooth muscle relaxation. For
sildenafil to exert its effect, it requires intact
NO-relaxing nerve fibers and an intact caver-
nosal epithelium. Sildenafil is not effective in
patients with vascular disease where NO pro-
duction is impaired, and has no effect in the
absence of sexual stimulation.
Sildenafil has high selectivity for PDE5. It
has 80- to more than 8500-fold selectivity to
PDE5 versus PDE1, PDE2, PDE3, and PDE4
(125). PDE6, the isoenzyme found in the ret-
ina, is closely related to PDE5. The selectivity
of sildenafil to PDE6 is one-tenth of that to
PDE5 (143). This explains the visual abnor-
malities observed with higher plasma levels of
s i l d e n d . In addition to the cavernosal tissue,
PDE5 is also found in lower concentrations in
other human tissues including platelets, vas-
cular smooth muscles, and skeletal muscles.
The inhibition of PDE5 in these tissues is be-
lieved to be responsible for the vascular side
effects of sildenafil.
6.2.5.2 Alprostadil. Alprostadil is chemi-
cally identical to the naturally occurring form
of prostaglandin El and acts similar to the en-
dogenous PGE. It induces erection by relax-
ation of the cavernosal smooth muscle and di-
lation of cavernosal arteries, which leads to
increased arterial inflow and decreased ve-
nous outflow. Alprostadil has various systemic
Lifestyle and Over-the-counter Drugs
effects including vasodilatation, inhibition of
platelet aggregation, and stimulation of intes-
tinal and uterine smooth muscles.
6.2.5.3 Yohimbine. Yohimbine is an a-ad-
renoreceptor antagonist, with high selectivity
to the presynaptic a2-adrenergic receptors. It
is generally believed that yohimbine exerts its
erectogenic effect by antagonizing the adren-
ergic inhibitory tone that suppresses erection.
The mechanism of action of yohimbine on
some sexual functions seems to be central, i.e.,
by inhibiting the presynaptic a2 receptors in
the brain (129). Published data from animal
experiments support the postulation that yo-
himbine's effects on sexual arousal and ejacu-
lation occur through a central mechanism,
because the drug can induce arousal even after
genital anesthesia (144) and it reverses
clonidine-induced inhibition of ejaculation
(145,146). However, the mechanism of action
of yohimbine on erectile function was not fully
understood and its site of action was not con-
clusively identified until it was demonstrated
few years ago that functional a2 receptors are
expressed in the human corpus cavernosum
(147). It is now believed that yohimbine exerts
its erectogenic action by blocking post-synap-
tic a2 receptors in the corpus cavernosum and
thus inhibits the contractility of the caver-
nosal tissue (128). In addition to its a-adren-
ergic effects, yohimbine exerts a stimulatory
action on the mood and may increase anxiety.
This effect is at least partially attributed to
yohimbine's central serotonergic activity
(148), although it has not been adequately
studied.
6.2.5.4 Other Agents. Phentolamine is a
nonselective a1- and a2-adrenoreceptor an-
tagonist. It is a weak erectogenic agent when
used alone as an intracavernosal injection.
Phentolamine is usually used in combination
with alprostadil andlor papaverine.
6.3 Chemistry and Structure-Activity
Relationships
Sildenafil(6) is a pyrazolopyrimidinone deriv-
ative. The pyazolopyrimidinone ring is essen-
tial for PDE5 inhibitory activity. It seems that
this ring structure mimics the guanosine base
of cGMP (149). Another ring system that has
been shown to produce potent and selective
PDES inhibitory activity is imidazoquinazoli-
6 Sexual Disorders
(6) Sildenafil Citrate
none (150). In the sildenafil series, the n-pro-
pyl substitution on position 3 of the pyazolopy-
rimidinone ring gives a more potent compound
compared with a 3-methyl analog. Intramolec-
ular hydrogen bonding of sildenafil and its an-
alogs is important for biological activity by
maintaining co-planarity between the phenyl
and purine ring (151). The 2'-alkoxy moiety on
the phenyl group serves this purpose by pro-
viding the oxygen lone pair for hydrogen bond-
ing with the pyrimidinone NH (149). The
alkoxy moiety also provides the requirement
for a small lipophilic substituent, which is im-
portant for activity. Replacement of the
alkoxy moiety with a hydrogen reduced PDE5
affinity by 200-fold (149). Hydroxy, nitro, and
sulphonamide derivatives also had lower af-
finity to PDE5. It has been demonstrated that
an open chain 2'-alkoxyl group serves the li-
pophilicity requirement better than a fused
ring system. An ether ring fused into the phe-
nyl moiety, although increasing the degree of
co-planarity, largely reduced the PDE5 inhib-
itory activity (151). An N-acylamido substitu-
tion at the 5'-position of the phenyl ring was
shown to enhance PDE5 inhibitory activity
(152). Sildenafil analogs with such a substitu-
ent were one to three times more potent than
sildenafil, based on in uitro PDE5 inhibitory
activity. The activity increased with increas-
ing the chain length of the N-acylamido moi-
ety. This substitution, however, decreased the
analogs' selectivity to PDE5 over PDE6 (152).
The sulfonamide substituent on position 5'
enhances the aqueous solubility of sildenafil
and also increases its affinity to cGMP (149).
Introduction of a carboxylic acid group to the
5'-sulfonamide moiety of the phenyl ring
greatly enhanced the in uitro PDE5 activity
(153), possibly caused by mimicking the phos-
phate group of cGMP.
Alprostadil (7) is a synthetic form of pros-
taglandin El (PGE,). It is chemically identical
(7) Alprostadil
to the naturally occurring PGE,. Prostaglan-
din El is an acidic lipid that is synthesized by
mammalian tissues from fatty acid precur-
sors.
Yohimbine (8)is a natural alkaloid found in
Rubaceae and related trees, mainly, from the
(8) Yohimbine
bark of the African tree Pasinystalia yohimbe,
and is also found in Rauwolfia Serpentina. It is
an indolalkylamine alkaloid with structural
similarity to reserpine. Chemically, it is known
as 17a-hydroxy-yohimban-16a-carboxylic acid
methylester. The commercial product con-
tains the hydrochloride salt of yohimbine.
6.4 History
The use of drugs for the treatment of impo-
tence and other sexual disorders is very old.
Yohimbine has been used in folk medicine by
men and women for various forms of sexual
dysfunction, mainly as an aphrodisiac for over
a century. In the United States, yohimbine hy-
drochloride has been marketed and prescribed
by doctors for more than 75 years (154). Be-

cause the mainstream medical use of yohim-
bine pre-dated the Food and Drug Act, the
drug did not go through the formal FDA re-
view and approval process, and hence, there
are no well-controlled clinical trials with a suf-
ficient number of patients to allow for conclu-
sive results on its efficacy and long-term
safety. The lack of conclusive clinical data and
the daily dosing schedule that has been prac-
ticed for decades, rather than a more conve-
nient on-demand use, are the main reasons for
the low level of interest in yohimbine by med-
ical practitioners. Needless to say, pharma-
ceutical companies lack the interest in con-
ducting expensive large-scale clinical trials of
yohimbine, because the ancient drug is non-
patentable.
Because of the absence of clinically proven
safe and effective pharmacotherapy for erec-
tile dysfunction, psychotherapy was until re-
cently the core treatment for most patients,
except for a few cases where a surgically cor-
rectable cause could be identified. The results
of psychotherapy have been disappointing
(155), and often times, the goal of psychother-
apy was to help men accept their sexual dys-
function rather than to correct it. The wide-
spread belief that erection failure is primarily
of psychological origin, and hence the accep-
tance of psychotherapy as the primary treat-
ment, continued until the early 1980s.
In 1982, a new era for erectile dysfunction
started when Virag (156) showed that penile
injection of a vasoactive amine-papaverine
(9)-could result in erection without sexual
(9) Papaverine
stimulation. In 1983, Brindley (157) reported
the clinical efficacy of cavernosal a-blockade in
the treatment of erectile dysfunction. As a re-
Lifestyle and Over-the-counter Drugs
sult of the extensive research on the mecha-
nism of erection that followed Virag's and
Brindley's pioneer work, it was established
that underlying organic causes are responsi-
ble for approximately 80% of persistent erec-
tile dysfunction cases (142). This resulted in
changing the primary treatment from psycho-
therapy to pharmacotherapy and stimulated
further research in this area. Intracavernosal
injections of papaverine and phentolamine
(10) became a common treatment for erectile
(10) Phentolamine
dysfunction in addition to vacuum/constric-
tion devices. As a result of the growing inter-
est and increased understanding of the condi-
tion, the U.S. National Institute of Health
(NIH) advocated the use of the term erectile
dysfunction (ED) rather than impotence in
1992 (158).
The first pharmacological agent for ED to
be approved by the FDA was intracavernosal
alprostadil. It was approved in June 1995 for
the treatment of erectile dysfunction caused
by neurogenic, vasculogenic, psychogenic, or
mixed etiology. Despite its clinical efficacy, the
high cost, and more importantly, fear of self
injection limited the use of intracavernosal
therapy. The search for a less invasive therapy
led to the development of a novel transure-
thral dosage form of alprostadil, which gained
FDA approval in 1997. This was still far from
the ideal therapy. The main disadvantages of
alprostadil therapy are loss of spontaneity, be-
cause erection occurs even without sexual
stimulation, and rapid onset of action, which
does not allow for discreet administration and
also contributes to the loss of spontaneity.
In 1985, the story of sildenafil discovery
(159) started when two chemists at Pfizer's
site in Kent, U.K. proposed to look for antihy-
pertensive and antianginal compounds that
 6 Sexual Disorders
would work by inhibiting phosphodiesterase
(PDE) and thus promoting the vasodilator ac-
tion of cGMP. The project started with the
usual literature review to identify a chemical
starting point. Zaprinast (111, a compound
(11) Zaprinast
that was developed as an antiallergic by May
and Baker (later part of Rhone-Poulenc Rorer,
which is now part of Aventis), was selected.
Zaprinast, which was never commercialized,
was not selective enough to PDE; however, it
provided a satisfactory starting point for the
Pfizer team. In their effort to modify the Zap-
rinast structure to make it more selective and
more potent, Pfizer chemists explored other
ring systems and varied the side-chain substi-
tutions. A total of about 1600 compounds were
synthesized. In 1989, sildenafil (UK92480)
was identified as a promising candidate based
on its in vitro selectivity to PDE and its po-
tency (159). Sildenafil had a more than 500-
fold affinity to PDE than the starting com-
pound (149). Phase I clinical trials of sildenafil
started in 1991. In 1992, the results of a lim-
ited phase I1 clinical study in patients with
severe coronary heart disease were disap-
pointing, but at the same time, the first report
of sildenafil's effect on erectile function came
from a parallel high dose phase I study. The
"side effect" of erection caught the team's in-
E terest; however, the decision to change the di-
; rection of the sildenafil development program
-
1
from cardiovascular to erectile dysfunction
came only after long deliberations (159). The
I discovery of nitric oxide and the understand-
I
ing of its role as a signaling molecule in the
1
I
early 1990s helped Pfizer's researchers to un-
derstand the mechanism of sildenafil's effect
on erection and therefore helped in making
1 that decision. The first phase I1 clinical trial
j for erectile dysfunction started in 1994 and
included 12 men with ED. Ten of 12 patients
showed improvement. The next trial, which
took place in 1994 and 1995, was an outpatient
trial. This was followed by a large open-label
multi-center clinical trial in which 225 pa-
tients were assessed for over 32 weeks. Eighty-
eight percent of patients reported improve-
ment, and over 90% expressed interest in
continuing the treatment. In total, the drug
was tested in about 4000 men, ages 19-87,
who suffered from erectile dysfunction of or-
ganic, psychogenic, or mixed origin, or with no
identified etiology (117). In all of the studies,
sildenafil exhibited superior efficacy over pla-
cebo. The NDA was submitted to the FDA in
September 1997, and sildenafil was approved
in March 1998 after a fast track review.
6.5 Future Trends
A few dozen potential products are in clinical
development for male and/or female sexual
disorders. Several of these potential products
are novel delivery systems of already mar-
keted compounds, such as two topical delivery
systems of alprostadil developed by NexMed
and Macro Chem, and a lyophilized liposomal
delivery system for urethral administration of
alprostadil, developed by Harvard Scientific.
New oral agents that target central and pe-
ripheral pathways of erection are expected t~
be introduced in the next few years. Some of
the older off-label agents are also in late
phases of clinical development or under FDA
review. An injectable formulation of phentol-
amine is being developed by Novartis, and
Senetek is developing a combination of
vasoactive intestinal peptide (VIP) and phen-
tolamine in an autoinjector. Trazodone is an-
other older drug marketed as an antidepres-
sant and is in clinical development for erectile
dysfunction. Trazodone (12) is a centrally act-
ing serotoninergic agonist with peripheral
sympatholytic activity. Studies indicate that it
0
(12) Trazodone

is mostly effective in patients with psycho-
genic etiology (160). Other serotonin agonists
are being tested by Vivus for premature ejac-
ulation and other sexual disorders.
Apomorphine (13) is a dopaminergic ago-
nist that acts at the paraventricular and su-
(13) Apomorphine
praoptic nuclei in the brain, but has low oral
bioavailability. TAP Pharmaceuticals is devel-
oping a sublingual formulation of apomorphine
for the treatment of erectile dysfunction. Re-
cently, TAP withdrew its NDA application of
the sublingual preparation after the FDA ad-
visory panel rejected the use of 4 mg of apo-
morphine in ED treatment because of safety
concerns. The company hopes to resubmit the
2-mg NDA application in October 2002. Nas-
tec is testing an intranasal formulation of apo-
morphine (1mg) for erectile dysfunction. The
lower dose will help reduce the side effects.
In addition, several new PDE5 inhibitors
are in various phases of clinical development.
The closest to marketing are ICOSEli Lilly's
IC-351 (Cialis) and Bayer's vardenafil. Both
drugs are expected to be approved by the FDA
in 2002. Other novel compounds include
didesmethylsibutramine (by Sepracor), which
is a single isomer of an active metabolite of
sibutramine-a norepinephrine and dopa-
mine reuptake inhibitor--and nitric oxide
NMI-870 (NitroMed), which is a nitric oxide-
enhanced compound of yohimbine for specific
delivery of NO to target tissue.
Melanotan I1 or PT-141 (Palatin Technolo-
gies) is a synthetic analog of melanocyte-stim-
ulating hormone (161). The peptide is cur-
rently under development as an intranasal
formulation for both male and female sexual
dysfunction. It is a non-selective melanocortin
receptor agonist, which in animals regulates
sex& behavior including penile erection, sex-
ual motivation, and in female rats, the secre-
Lifestyle and Over-the-counter Drugs
tion of sexual attractants from the preputial
gland. Melanotan, which was originally dis-
covered and synthesized by researchers at the
University of Arizona, started as a suntanning
agent. Spontaneous erection reported in a tan-
ning clinical study led to the initiation of a
joined development program between Univer-
sity of Arizona and Palatin Technologies. Re-
sults from phase IIA studies were very posi-
tive, with response rate greater than 80% and
good safety profile (162). Phase I11 trials for
erectile dysfunction are expected to start in
2003. Topical testosterone (gel formulations
from Solvay and Cellegy, and a patch from
Watson) is being tested for reduced sexual de-
sire in women. Bupropion was shown to im-
prove sexual desire in a pilot clinical study,
and the indication is likely to be pursued.
7 SMOKING CESSATION AGENTS
Although smoking cessation is a lifestyle deci-
sion, the ability to quit is usually hindered by
nicotine chemical dependence. According to
the Surgeon General report on smoking in
2000 (163), "Tobacco dependence is in fact
best viewed as a chronic disease with remis-
sion and relapse. Even though both minimal
and intensive interventions increase smoking
cessation, most people who quit smoking with
the aid of such interventions will eventually
relapse." It is estimated that 24.7% of all
adults who lived in the United States in 1997
were smokers (164). This is only slightly lower
than the 25.5% prevalence in 1994 (165). Sta-
tistics show that over 70% of smokers express
a strong desire to quit and about 20% actually
try to stop smoking. However, only 3-5% of
smokers who attempt to quit smoking on their
own achieve a successful abstinence after 1
year (165, 166). The agency for Health Care
Policy and Research (AHCPR) in its Clinical
Practice Guidelines on Smoking Cessation re-
leased in April 1996 (The U.S. Public Health
Service practice guidelines) (167) state that
every patient attempting to stop smoking
should be treated with pharmacotherapy.
Pharmacological intervention is often part of a
multicomponent therapy that also includes
one or more non-pharmacological compo-
nents, mainly psychosocial therapy (behav-
 7 Smoking Cessation Agents
ioral therapy). The AHCPR found that smok-
ing cessation interventions by healthcare
providers doubled the success rate of smoking
cessation (167). When multiple types of pro-
viders (e.g., clinicians and psychotherapists)
deliver these interventions, the likelihood of
smoking cessation increases by a factor of
four. The panel also found that nicotine re-
placement therapy (gum and patches) is effi-
cacious regardless of the use of adjuvant treat-
ment (psychosocial interventions), but its
efficacy is increased when used with adjuvant
interventions.
Treating tobacco dependence is one of the
most cost-effective preventive measures. It is
estimated that smoking cessation is more cost-
effective than several common clinical preven-
tive services such as screening for cervical,
breast, and colon cancer, treatment of mild-to-
moderate high blood pressure, and treatment
of high cholesterol (163).
7.1 Clinical Use
7.1.1 Current Drugs. The FDA-approved
smoking cessation medications include nico-
tine replacement therapy (NRT) available in
several delivery systems and one non-nicotine
drug. Only nicotine gum and nicotine patches
are available over-the-counter. Table 9.9 lists
smoking cessation medications that are ap-
proved for clinical use in the United States.
The dose of NRT varies depending on the de-
livery system and the current usage level of
tobacco by the patient. Bupropion therapy is
usually started 1 week before quitting smok-
ing as 150-mg slow-release tablets twice a day.
j The usual length of treatment of smoking ces-
1
sation therapy is 6-12 weeks. Because all the
approved smoking cessation agents are almost
1 equally effective and safe (1681, the choice of
j/ treatment usually depends on the patient
preference. Bupropion is particularly useful
i for smokers who had unsuccessful attempts
1 with NRT. It is also useful for smokers who
are concerned about possible weight gain after
quitting. Bupropion was shown to attenuate
weight gain after smoking cessation; however,
the benefits are not sustainable once the treat-
ment is discontinued (169,170). Although the
OTC labels recommend against using nicotine
gum or patches in combination with other nic-
i
!
otine products, there is evidence that combin-
ing a nicotine patch with 2-mg gum ad libitum
for high craving instances may be advanta-
geous (171). The results, however, were not
sustainable in clinical trials after 12 months
(172, 173). Using the nasal spray along with
the patch for severe craving also increased the
success rate (174). The combination of bupro-
pion and nicotine patch was found to be more
effective than either treatment alone, al-
though the difference between the combina-
tion and bupropion alone was not statistically
significant (175, 176).
7.1.2 Adverse Effects. The main adverse
effect of nicotine is psychoactive substance de-
pendence. Acute and chronic tolerance to nic-
otine effects on the brain function and activity
lead to increased nicotine consumption through
smoking (177, 178). Acute tolerance to nico-
tine can develop in less than 1 h, but develops
at different rates for different physiological ef-
fects. Because of the highly addictive effects of
nicotine, smoking cessation results in nicotine
withdrawal syndrome. The symptoms can
start as early as 2 h after the last nicotine
consumption and reach a peak in 24-48 h af-
ter smoking cessation. The symptoms include,
in addition to tobacco craving, depression, in- .
somnia, irritability, nervousness, restless-
ness, difficulty concentrating, anxiety, drows-
iness, sleep disturbances, decreased heart
rate, and increased appetite (179, 180). Side
effects of excess nicotine consumption include
nausea, vomiting, abdominal pain, diarrhea,
flushing, dizziness, disturbed hearing and vi-
sion, weakness, confusion, and palpitation.
Side effects specific to nicotine delivery sys-
tems are mainly caused by local irritation at
the site of administration. For example, nau-
sea, indigestion, sore gums, and mouth ulcer-
ation may occur when using the gum. The
patches may cause skin irritation, which is
characterized by erythema, pruritus, edema,
and rarely, vesicles. The nasal spray may
cause irritation of the nasal mucosa, sneezing,
coughing, and lacrimation, although tolerance
to these effects develops rapidly with contin-
ued use. Side effects from the inhaler include
mild mouth and throat irritation and cough-
ing (181).
 Table 9.9 Current Smoking Cessation Agents on the U.S. Market
Current
Brand Delivery Regulatory
Generic Name Name System Originator Strength Daily Dose Status
Bupropion Zyban SR tablet Glaxo Wellcome 150 mg 300 mg Prescription
hydrochloride
Nicotine Habitrol Transdermal Novartis 21,14,7 mg 1 patch OTC
system
e
N Nicotine Nicotrol Transdermal CygnusPharmacia 15 mg 1 patch OTC
system
Nicotine Nicotrol Inhalation Parke-Davis 10 mgICartridge 6-16 Prescription
inhaler system (4 mg delivered) Cartridges
Nicotine Nicotrol NS Nasal spray Parke-Davis 10 mg/mL (0.5 Up to 40 mg Prescription
mglspray) (80 sprays)
Nicotine Nicorette Nicotine- Lakeside 2 mg, 4 mg Up to 24 OTC
polacrilex polacrilex Pharmaceuticals pieces
gum (Merrell Dow)
, 7 Smoking Cessation Agents
Bupropion is a well-tolerated antidepres-
sant. It is non-sedating and lacks the cardio-
! vascular and anticholinergic side effects of tri-
1
tI
cyclic antidepressants. Bupropion's most
commonly observed adverse effects are insom-
nia and dry mouth. The drug is also known to
be associated with a low rate of seizures; how-
ever, no seizure incidents were reported in the
smoking cessation clinical trials. Other less
frequently occurring side effects include ner-
vous system disturbances (mainly tremor) and
skin rashes (170, 182).
7.1.3 Pharmacokinetics. Nicotine is readily
available by inhalation after tobacco smoking.
Inhalation is the fastest route for nicotine ab-
sorption and results in the highest bioavail-
ability. The bioavailability from cigarettes in
smokers who inhale is about 90% (183). These
smokers attain a maximum blood level almost
instantaneously. Nicotine, a small lipophilic
molecule, is also rapidly absorbed through
skin and the mucous membranes. It is a basic
compound, and therefore, is absorbed in a pH-
dependent manner. Absorption through the
oral mucosa seems to be slower than the nasal
route (possibly because of the pH effect). Cig-
arette smoke is mildly acidic (pH 5.3) and,
therefore, only little absorption occurs in the
buccal cavity (about 10%). However, in the al-
kaline smoke of pipes and cigars (pH 8.51, nic-
otine is absorbed rapidly through the oral mu-
cosa (183). Nicotine penetrates the blood-
brain barrier and reaches the central nervous
system rapidly, with maximum brain concen-
tration reached within a few seconds after cig-
arette smoking (182). In general, absorption
from nicotine-replacement drug delivery sys-
tems is controlled by the release rate from the
vehicle. The nasal spray provides the fastest
release and hence fastest absorption of nico-
tine, and therefore has the advantage of rapid
alleviation of nicotine withdrawal symptoms.
The maximum nicotine concentration is
reached in 4-15 min after administration of
nicotine nasal spray, which is still slower than
the almost instantaneous peak after smoking
tobacco. Nicotine absorption from the nicotine
polacrilex gum is slower because of a slower
release rate from the delivery system and
slower absorption through the oral mucosa.
When used properly, each piece of the gum
provides continued absorption for approxi-
mately 30 min. Although the inhaler has the
advantage of mimicking the act of smoking,
absorption rate is comparable with Nicorette
gum and slower than that from the nasal
spray. Nicotine absorption from the "inhaler"
occurs through the buccal mucosa rather than
the lungs. Nicotine patches, extended release
transdermal delivery systems, maintains the
most sustained blood level and therefore re-
quires less frequent dosing.
Nicotine bioavailability also varies with the
delivery system. The reported bioavailability
from the nasal spray and transdermal patch is
53% and 82%, respectively (93, 184). In nico-
tine polacrilex gum, nicotine is bound to an ion
exchange resin and is released only by chew-
ing. Nicotine bioavailability, therefore, is de-
pendent on the vigor, rapidity, and duration of
chewing. Of the 10 mg in each cartridge of a
nicotine inhaler, only 4 mg is actually deliv-
ered from the device to the oral mucosa and is
available for absorption.
Nicotine has low plasma protein binding
(4%) and a large volume of distribution (2-3
L/kg). It is eliminated mainly by hepatic me-
tabolism, although some metabolism occurs in
the lungs and kidneys. The main metabolites
are cotinine (15% of the dose) and trans-3-hy-
droxycotinine (45% of the dose). Only 10% of .
the absorbed dose is excreted unchanged in
urine. In healthy adult smokers, nicotine has
an apparent elimination half-life of 1-2 h and
the average plasma clearance is 1.2 h (93).
Bupropion oral bioavailability in humans
has not been determined because the drug was
never administered intravenously to humans.
The relative oral bioavailability in rats and
dogs ranges between 5% and 20%. In healthy
volunteers, peak plasma concentration is
reached approximately 3 h after administra-
tion of the sustained release tablet (93). Based
on in vitro protein binding data, bupropion is
84% bound to plasma proteins. It is widely dis-
tributed to tissues and has an apparent
steady-state volume of distribution (Vss/F) of
approximately 2000 L. Bupropion is exten-
sively metabolized by oxidation and reduction
to at least six metabolites with only 0.5% of a
bupropion oral dose excreted unchanged in
urine. The major metabolite in urine is a gly-
cine conjugate of metachlorobenzoic acid,

which is formed by oxidation of the bupropion
side-chain followed by glycine conjugation.
Three active metabolites have been identified:
hydroxybupropion and the two amino-alcohol
isomers threohydrobupropion and erythrohy-
drobupropion (185). These metabolites are
formed through hydroxylation of the tert-bu-
tyl group of bupropion and/or reduction of the
carbonyl group. The potency of the active me-
tabolites has not been assessed in humans.
However it is suggested that hydroxybupro-
pion has a primarily noradrenergic effect,
whereas threohydrobupropion has some dopa-
minergic activity and minor noradrenergic ef-
fect (186). The CSF concentrations of hy-
droxybupropion and erythrohydrobupropion
are 6 times greater than the parent drug,
whereas threohydrobupropion CSF concen-
tration is 40 times that of the parent drug
(185). In vitro data suggest that cytochrome
P450IIB6 (CYP2B6) is the principal enzyme
involved in the formation of hydroxybupro-
pion, whereas P450 isoenzymes are not in-
volved in the formation of threohydrobupro-
pion. The mean apparent clearance (CZIF)of
bupropion ranges between 135 and 209 L/h.
The mean apparent elimination half-life of bu-
propion after administration of the sustained
release tablet is approximately 21 h. The elim-
ination half-life from the immediate release
formulation is 11-14 h. The main elimination
half-lives of the active metabolites are 20, 37,
and 33 h for hydroxybupropion, threohydro-
bupropion, and erythrohydrobupropion, re-
spectively (93).
7.2 Physiology and Pharmacology
7.2.1 Pharmacological Action of Nicotine.
Nicotine binds selectively to the nicotinic
receptors that are present in the adrenal me-
dulla, brain, autonomic ganglia, and neuro-
muscular junctions. It causes the release of
several neurotransmitters and hormones such
as acetylcholine, norepinephrine, dopamine,
serotonin, arginine vasopressin, p-endorphin,
adrenocorticotropic hormone, and cortisol
(187). This neuro-regulatory effect of nicotine
is dose-dependent and occurs as plasma nico-
tine level rises when tobacco is smoked. The
neurotransmitters released in the brain medi-
Lifestyle and Over-the-counter Drugs
ate the behavior modulating effects and posi-
tive reinforcing effects of nicotine and other
habit-forming drugs.
7.2.1.1 Neurological Basis of Nicotine De-
pendence. In the brain, nicotine exerts a mul-
titude of psychological and behavioral effects.
At low doses, it exerts a predominantly stimu-
lating effect, which occurs in the cortex
through locus ceruleus and is mediated by nor-
epinephrine. At high doses, a dopaminergic re-
ward effect predominates (184, 188). The re-
ward and positive reinforcement effects of
nicotine are responsible for the drug-seeking
behavior in tobacco smokers. A common effect
of many drugs of abuse, as well as natural re-
wards (e.g., food, sex), is the elevation of extra-
cellular dopamine levels (189). The mesolim-
bic-dopaminergic neurons in the midbrain are
thought to be the final common pathway for
reward, and the reward effect occurs as a re-
sult of the elevated dopamine level. Although
there have been some recent challenges to the
dopamine reward theory (190-193), it is still
unequivocally agreed that dopamine plays a
crucial role in the reward system and drug-
seeking habit formation. An alternative view
to the classical dopaminergic reward theory is
that the dopaminergic-neuron activation
functions as a learning signal (189, 194), and
that the neuroadaptive changes (up-regula-
tion) of the dopaminergic neurons with the
chronic use of tobacco (or other drugs of
abuse) might result in the generation of a def-
icit state that enhances drug craving and
hence drug-seeking behavior (189). Other
neurotransmitters such as norepinephrine
and 5-HT have been implicated in the reward
and positive reinforcement effects of nicotine.
Nicotine is an agonist to the neuronal nic-
otinic acetylcholine receptors (nAChR).These
receptors are the likely site at which nicotine
exerts its central actions. Evidence supports
the involvement of the central nAChR in neu-
rotransmitter release (195), and a large body
of evidence indicates that dopamine release
from dopaminergic neurons is mediated by ac-
tivation of the brain nAChRs (196). It has also
been shown that both nicotinic receptors and
muscarinic receptors in the ventral tegmental
area (VTA)activate the dopaminergic neurons
and thus play a role in the reward effect of
nicotine (197). In addition, animal data sug-
7 Smoking Cessation Agents
gest an involvement of the 5-HT (2C) receptor
in mediating the mesolimbic-dopaminergic
system (198).
These positive reinforcement effects of nic-
otine include anxiolytic effect, antinocicep-
tive/analgesic effects, enhanced vigilance, and
improved cognitive function. Tolerance to
many of these effects occurs rapidly, leading to
substance dependence (199, 200). In animal
studies, a single pretreatment with nicotine
results in acute tolerance to the subsequent
dose (201,202). The role of norepinephrine in
the positive reinforcement effects of nicotine
is not clear, but it has been suggested that
nicotine's effects on concentration and atten-
tion are mediated by a noradrenergic mecha-
nism (182). The noradrenergic system is also
implicated in mediating the effects of nicotine
withdrawal. 5-HT receptors in the dorsal ra-
phe nucleus (DRN) are implicated in the anxi-
olytic effect of nicotine (203). Changes to these
receptors mediate the development of toler-
ance to the anxiolytic effect and hence the anx-
iogenic response during nicotine withdrawal
(203). The antinociceptive/analgesic effect of
nicotine is believed to be mediated by a cholin-
ergic pathway through the neuronal nicotinic
acetylcholine receptors (204, 205). 5-HT re-
ceptors are also implicated in the antinocicep-
tive effect of nicotine, possibly through an
interaction between the nicotinic and seroto-
nergic systems (206). Acetylcholine is known
to play a crucial role in nicotine's effects on the
cognitive function.
7.2.1.2 Peripheral Pharmacological Actions
of Nicotine. Nicotine effects on the cardiovas-
cular system include tachycardia and periph-
eral vasoconstriction, which leads to elevated
blood pressure. Because the cardiovascular ef-
fects are mainly caused by elevated levels of
catecholamines and cortisol, tolerance to
these effects does not occur. Other pharmaco-
logical actions of nicotine include increased
gastrointestinal motility caused by parasym-
pathetic ganglionic stimulation and skeletal
muscle contraction caused by the effect on nic-
otinic receptors in the neuromuscular junc-
tion (184).
7.2.2 Bupropion Mechanism of Action. Bu-
propion is a monocyclic non-MA0 inhibitor
antidepressant with both dopaminergic and
noradrenergic activities. It is a weak inhibitor
of dopamine, norepinephrine, and serotonin
reuptake (207), but it has a greater effect on
the neuronal reuptake of catecholamines than
of serotonin. Some studies suggest that bupro-
pion is entirely selective for catecholamines
and is devoid of any serotonergic activity
(185). Bupropion's effectiveness as a smoking
cessation agent is not related to its antidepres-
sant effect. The drug is equally effective in
smokers with current depression, past depres-
sion, or no depression (168). The mechanism
by which bupropion enhances the ability for
smoking cessation is believed to be related to
its dopaminergic and noradrenergic activity
(186). In addition, it has been shown recently
that bupropion is a potent inhibitor of central
nAChRs and that it blocks nicotine activation
of these receptors in a dose-dependent non-
competitive fashion (208). This suggests that
the nicotinic antagonist effect contributes to
bupropion mechanism of action in smoking
cessation.
7.3 Chemistry and Structure-Activity
Relationships
Nicotine (14) [S-3-(1-methyl-2-pyrrolidinyl)
pyridine] is the main alkaloid in tobacco. It is a
tertiary amine composed of a pyridine and a
(14) Nicotine
pyrrolidine ring. Nicotine exists in two iso-
meric forms, but tobacco contains only the
levorotatory isomer, which is the more phar-
macologically active form.
7.3.1 Nicotinic Acetylcholine Receptors. Ni-
cotinic acetylcholine receptors are ligand-
gated ion channels whose opening is con-
trolled by acetylcholine and nicotine agonists
(196,209,210). They are transmembrane pro-
tein structures. Each receptor consists of five
subunits. The structure is inserted in the
plasma membrane with an aqueous channel in
456
the center. The subunits have common gen-
eral structure that comprises the following:
0 A large extracellular N-terminal that forms
the bulk of the acetylcholine-binding site
0 Four putative transmembrane hydrophobic
regions that form the channel
0 An intracellular loop joining the third and
fourth transmembrane domains: this is a
very important structure for the regulation
of receptor function
An extracellular C-terminus
In the CNS, the nAChRs are located mainly
at the presynaptic nerve terminals where they
modulate the synaptic activity by regulating
the neurotransmitter release. Multiple popu-
lations of nAChRs exist, with a largely diverse
subunit composition.
The nAChR subunits are grouped into two
main types: ligand-binding subunits (a sub-
units) and structural subunits (p subunits).
Several types of the a and p subunits have
been identified. The subunits that are present
in the neuronal nAChRs are a2-a9 and p2-p4.
The pharmacological properties of the nAChR
subtypes largely depend on their subunit com-
position. For example, the analgesic effect is
believed to be mediated by the a4p2 subtype,
whereas the dopamine release from dopami-
nergic neurons in the brain is controlled in
-part by another a4-containing - subtype and
- -
possibly by an a6-containing subtype.
Since the discovery of the neuronal
nAChRs, there has been a rapidly growing in-
terest in designing drugs that are selective to
neuronal nAChR subtypes and hence can spe-
cifically target diseases or clinical conditions
such as smoking addiction, pain, anxiety, at-
tention deficit, Alzheimer disease, Parkinson
disease, and other cognitive and neurodegen-
erative disorders.
7 - 3 2 Structural Requirements for nAChR
Ligands. Schmitt (211) summarized the gen-
era1 structural requirements for binding at
the a4P2 and a 7 receptors (the most abun-
dant nAChRs subtypes in the CNS) as fol-
lows.
Lifestyle and Over-the-counter Drugs
1. A cationic center, preferably a basic or
quaternized nitrogen, is required. In nico-
tine, this requirement is satisfied by the
pyrrolidine nitrogen.
2. A hydrogen bond acceptor (HBA) and/or
T-electron rich moiety is favored. This is
satisfied by a less basic nitrogen such as the
pyridine nitrogen of nicotine or a carbonyl
oxygen (e.g., the carbonyl oxygen of
acetylcholine).
3. The receptor favors a relative separation
between the cationic center and the
H-bond acceptor or T-electron moiety of
4-8 A.
4. The receptors exhibit the tendency toward
stereospecific interaction.
5. The receptor may prefer some degree of
cation-HBNT coplanarity.
7.3.3 SAR of Bupropion and Its Analogs.
Bupropion (15), also known as amfebuta-
mone, is 1-(3-chloropheny1)-2-[(l,l-dimethyl-
(15) Bupropion
ethy1)aminol-1-propane.It is a trimethylated
monocyclic phenylaminoketone and is struc-
turally unrelated to the tricyclic antidepres-
sants or MA0 inhibitors. Commercially avail-
able bupropion is a racemic mixture. The two
enantiomers were synthesized and assayed for
their potencies as inhibitors of biogenic amine
uptake into nerve endings obtained from
mouse brain (212). No significant difference
was found. However, the relative pharmaco-
logical activities and pharmacokinetics of the
two enantiomers have not been studied. Bu-
propion has a novel chemical structure among
antidepressants. The absence of polycyclic
rings and presence of more common func-
tional groups usually found on tranquilizers
contribute to the lack of marked side effects
I 7 Smoking Cessation Agents
usually seen with polycyclic antidepressants
(213). Bupropion was a designed antidepres-
sant. A series of compounds that included ami-
noketones and aminoalcohols was designed
and screened for antidepressant activity by
Glaxo chemists. Structure-activity relation-
ships of a series of bupropion analogs were
investigated (213). The strong electron with-
drawing effect of the chloro substituent on the
aromatic ring in bupropion is believed to be
responsible for the lack of CNS stimulant ef-
fect. The a-ketone moiety contributes to the
metabolic fate of the drug. It prevents the for-
mation of a chloro-monoarylalkylamine me-
tabolite, which would possibly be a CNS stim-
ulant. The use of a tertiary butyl substituent
as the alkyl group on the nitrogen atom was
designed to diminish the N-dealkylation and
hence prevent the formation of metabolites
with sympathomimetic side effects. The meta-
orientation of the two substituents on the ar-
omatic ring was also a designed feature. The
ortho position could have high steric hin-
drance, and the para position would result in
facile displacement and para-hydroxylation,
which in turn would result in rapid elimina-
tion of the drug.
7.4 History
Early research on smoking cessation therapy
started in the 1930s. The first experimental
medication was lobeline, an alkaloid with
physiological actions similar to nicotine. Lobe-
line and other early non-nicotine drugs failed
to show any benefits beyond those seen with
placebo (170). As a result of poor efficacy of
these experimental drugs, researchers started
focusing on nicotine. The first U.S. Surgeon
General's report on smoking, which was re-
leased in 1964 (214), stimulated the research
efforts on nicotine's pharmacological and
physiological effects and its role as the main
addictive substance in tobacco. The first gen-
eration of smoking cessation medication was
nicotine polacrilex gum. It was approved by
the U.S. Food and Drug Administration in
1984. The second generation was the nicotine
transdermal delivery system or patches, the
first of which was approved in 1991. Three
other patches followed by 1992. Nicotine nasal
spray, approved in 1996, and the nicotine in-
haler, approved in 1997, were the third gener-
ation.
Although NRT has consistently demon-
strated better efficacy than placebo, the suc-
cess rate is still relatively low (30% at the end
of treatment and 20% 6-12 months later)
(170). This, in addition to increased knowl-
edge on the neurochemical basis of nicotine
dependence, renewed the interest in non-nic-
otine therapy for treatment of nicotine depen-
dence. Clinical trials on clonidine as an exper-
imental non-nicotine therapy in the 1980s led
to an interesting and unexpected finding of a
strong association between nicotine depen-
dence and depression. Although the study ex-
cluded smokers with evidence of depression,
analysis of the participants' data showed that
60% had an episode of major depression in the
past. Furthermore, depressed mood was one of
the symptoms reported frequently by partici-
pants in the first week after quitting (215).
These findings suggested that antidepressants
might be useful in smoking cessation therapy.
Researchers started looking - into marketed an-
tidepressants for the indication. Ferry et al.
(216) selected the antidepressant bupropion
because of its dopaminergic activity, because
it has been long believed that the reward sys-
tem is activated through a dopaminergic
mechanism. Bupropion, already marketed un-
der the brand names Wellbutrin and Wellbu-
trin. SR tablets, was first launched in the
United States as an antidepressant in 1986,
but was voluntarily withdrawn the following
year because of concerns over seizure side ef-
fects. Glaxo Wellcome was subsequently able
to demonstrate to the FDA that the level of
seizures in bulimic patients was acceptable,
and therefore, the antidepressant was re-
launched in 1989. The outcome of the first
open-label smoking cessation clinical trial was
very positive. It was then followed by three
double-blind placebo-controlled studies in
non-depressed smokers (a dose-response
study, nicotine patch comparative study, and a
long-term maintenance trial). The results
from these studies demonstrated significant
increase in quit rate over placebo for the 150-
and 300-mg doses (182). This subsequently led
to U.S. Food and Drug Administration ap-
proval of bupropion sustained release tablets

as the first non-nicotine treatment for nico-
tine dependence in 1997. Glaxo Wellcome
launched the product under the brand name
Zyban for the new indication.
7.5 Current and Future Trends
Because of the limited success of NRT, smok-
ing cessation research efforts are focusing
more on non-nicotine medications. Several an-
tidepressant and anxiolytic agents, as well as
adrenergic and noradrenergic agents, were
tested recently or are in clinical trials for this
indication (170). There is emerging evidence
that antidepressants and anxiolytics may be
particularly helpful for subpopulations of
smokers (i.e., those with mood disturbances).
With the current trends in the smokers' pop-
ulation, anxiolytic and antidepressants will
become increasingly important.
It is speculated that the following emerging
trends will have an effect on the development
of smoking cessation therapies.
0 Smoking is becoming more recognized as
drug dependence, and therefore, smoking
cessation therapy will be viewed not only as
a cancer and cardiovascular disease preven-
tive measure, but also as a treatment for a
psychological disorder (drug dependence).
0 Current trends indicate that the population
of smokers is changing. Future smokers are
likely to be those who are more poor and
uneducated as well as those with psychiat-
ric/alcohoVdrug abuse disorders andlor
heavy nicotine dependence (217).
With the high cost-efficacy of smoking ces-
sation therapy, it is expected to eventually
become reimbursable (163,217).
0 More government regulations against to-
bacco products and tobacco advertisement.
Efforts of the government agencies will be
targeted at preventing the onset of smoking,
especially in young people, and protection of
nonsmokers (163).
0 Newer NRTs (nicotine inhaler and nasal
spray) will be switched to OTC market.
0 More use of NRTs and non-nicotine therapy
combinations to enhance the outcome.
In addition, with the increased knowledge
of the neurochemistry of substance depen-
Lifestyle and Over-the-counter Drugs
dence, it is anticipated that the next genera-
tion of smoking cessation agents will be novel
compounds designed to target neurotransmit-
ters and receptors that mediate the reward
system responsible for habit formation or to
target the neuroadaptive mechanism respon- -
-
sible for substance de~endenceand with-
drawal symptoms. Several compounds based
on these novel mechanisms are in the pipe-
lines of pharmaceutical companies. Nicotine
-
replacement
- is still an active area of research.
Several novel transmucosal drug delivery sys-
tems and improved second-generation trans-
dermal patches are in clinical development.
Oral nicotine combined with a bioavailabilitv "
enhancer is also undergoing clinical trials
(218). An antismoking vaccine being devel-
oped by Xenova (219) has shown successful
results in clinical trials. Table 9.10 lists some
of the compounds in various stages of develop-
ment as antismoking therapeutic agents (116,
220).
8 SUNSCREENS
Most people are at least occasionally exposed
to the sun for extended periods of time, either
as a result of their lifestyle and recreational
activities or as a normal part of their jobs. The
main adverse effects to exposure to the sun-'
light are sunburn, photoaging, and skin can-
cer. Skin cancer is the most common type of
cancer, accounting for almost 40%of all malig-
nancies (221). According to the American Can-
cer Society, overl.3 million people will be di-
agnosed with nonmelanoma skin cancer in the
United States each year, and 53,600 people
will be diagnosed with skin melanoma (221,
222). Approximately 9600 people will die from
various types of skin cancer, with the majority
dying from malignant melanoma. Unpro-
tected long-term exposure to sunlight is
blamed for about 90% of skin cancer cases.
Ultraviolet radiation (UVR) is believed to be
responsible for most of the harmful effects of
sunlight.
The UV spectrum is divided into three
bands: UVA, UVB,and UVC. The UVA band is
the longest wavelength. It ranges from 320 to
400 nm. It is further divided into two subsets:
UVA I (340-400 nm) and UVA I1 (320-340
1 8 Sunscreens
I
I
Table 9.10 Compounds in Development for Smoking Cessation
or Tobacco Addiction Prevention
Generic NameLaboratory
Code Originator Chemical Class Mechanism of Action
Methoxsalen (with oral ICNIToronto University Furocoumarin Cytochrome P450 2A6
nicotine) inhibitor, inhibits
first pass effect of
oral nicotine
Mecamylamine (transdermal Elan Bicycloheptanamine Nicotine antagonista
in combination with
nicotine)
NS-2359 NeuroSearch Unknown Mixed monoamine
(Denmark) reuptake inhibitor
GW 320659 (1555U88) GlaxoSmithkline Phenyl morpholinol Catecholamine
selective reuptake
inhibitor
LY 354740 Lilly Bicyclohexane Group I1 metabotropic
dicarboxylic acid glutamate receptor
agonist (glutamate
release inhibitor)
CP 526555 Pfizer Unknown Nicotinic partial
agonist
SR 141716 Sanofi-Synthelabo Unknown Central cannabinoid
receptor antagonist
Cotinine (NIH 10498) LecTec Pyrrolidinyl pyridine Nicotinic partial
(nicotine agonist
metabolite)
GW 468816 GlaxoSmithkline Unknown Glycine receptor
antagonist
BP-897 Bioproject (France) Naphthalene Dopamine D3 receptor
carboxamide agonist
derivative
TA-NIC Xenova/ImmuLogic Nicotine conjugated to Nicotine vaccine
a carrier protein (prophylaxis)
Nabi-NicVax Nabi Derivatized nicotine Nicotine vaccine
on a carrier protein (prophylaxis)
Hydroxy bupropion Sepracor Phenyl aminoketone Noradrenaline
(R-isomer of reuptake inhibitor
bupropion)
CMI 477 Millennium azabicycloheptane Nicotinic partial
agonist
LY 426965 Lilly Piperazinyl 5HT 1A antagonist
phenylbutanone
SSR 591813 Sanofi-Synthelabo Unknown Nicotinic partial
agonist
"Previously marketed as a hypertension treatment, but discontinued as in 1996.

nm). UVA is more abundant in the solar spec-
trum than UVB (15-20 times more than UVB)
(223) but is less potent in causing erythema or
sunburn (223). Because sunburn is the most
visible UV-induced skin damage, UVB used to
be blamed for most of the destructive effects of
UVR.However, it is now believed that UVA,
particularly UVA 11,is at least as damaging as
UVB. It has been shown that UVA I1 can cause
the same molecular effects (i.e., direct DNA
damage) as UVB (224). UVA penetrates
deeper into the skin and is believed to cause
more damage to the dermis layer (225). More-
over, UVA is the band where most of the pho-
460 Lifestyle and Over-the-counter Drugs

Table 9.11 Sunscreen Agents Approved for OTC Marketing in the United States
Absorbance Approved
Sunscreen agent Class Structure range (nm) concentration
Arninobenzoic acid ABA
(ABA)
Avobenzone Dibenzoylmethane
Cinoxate Cinnamates
Dioxybenzone Benzophenones
(benzophenone-8)
Homosalate Salicylates
Menthyl anthranilate Anthranilates
Octocrylene Cinnamates
Octyl methoxycinnamate Cinnamates
(ethylhexyl-p-
methoxy-cinnamate)
Octyl salicylate (2- Salicylates
ethylhexyl salicylate)
Oxybenzone Benzophenones
(benzophenone-3)
Padimate 0 Aminobenzoate
Phenylbenzimidazole Miscellaneous
sulfonic acid
Sulisobenzone Benzophenones
(benzophenone-4)
Titanium dioxide Physical agent
Triethanolamine Salicylates
salicylate (trolamine
salicylate)
Zinc oxide Physical agent

tosensitizing chemicals exert their effect, es-
pecially above 360 nm (in the UVA I range).
The UVB band, which ranges from 290 to 320
nm, is the most potent radiation in producing
erythema. It is also considered the radiation pri-
m k l y responsible for skin cancer, although re-
cent evidence shows that UVA is also carcino-
genic. The intensity of UVB radiation is higher
in the late morning and early afternoon. On the
positive side, UVB is the radiation responsible
for vitamin D, synthesis in skin.
UVC is the shortest wavelength of the UVA
radiation (200-290 nm). It is also known as
the germicidal radiation. Most of UVC is
screened out by the ozone layer before it
reaches the earth's surface. If it reaches the
skin, W C can cause some erythema; however,
most of it is absorbed by the stratum corneum.
8.1 Clinical Use
8.1.1 Current Drugs. Most sunscreens cur-
rently in use are compounds that have high
absorbance or reflectance throughout the en-
tire W B range, part of the UVA range, and in
some instances infrared wavelengths. They
are divided into two main groups: chemical
(organic) and physical (particulate) agents.
Physical sunscreening is the only way to block
radiation across the entire spectrum (UVB,
W A , visible, and infrared) (226). However, in
practice, combined chemical sunscreens or
combinations of physical and chemical agents
provide higher levels of protection than phys-
ical sunscreening alone, because of the limited
concentration of physical sunscreens that can
be incorporated in a formulation without
causing visible whitening when applied to the
skin. Table 9.11 lists the compounds consid-
ered by the FDA as safe and effective sun-
screens for the OTC market. The lists of ap-
proved sunscreens in Europe and Australia
are much longer, mainly because of the sim-
pler approval process for new sunscreens that
is implemented in those countries. Examples
8 Sunscreens
Table 9.12 Examples of Sunscreening Agents Approved for Use in Europe and Australia,
but Not Available in the United States
Sunscreen agent Class
Isoamyl methoxycinamate Cinnamate
3-methylbenzylidene camphor Camphor
4methylbenzylidene camphor Camphor
Octyl triazone Phenol
Bis-ethylhexyloxyphenol
methoxyphenyl triazine Phenol
Drometrizole trisiloxane Phenol
Ecamsule Camphor
of the sunscreening agents that are not ap-
proved for use in the United States but are
approved by the regulatory agencies in the Eu-
ropean Union and Australia are listed in Table
9.12. Comparison of sunscreening agents
available in Europe, the United States, Aus-
tralia, and Japan can be found in Hayden et al.
(227) and Steinberg (228).
In addition to the 16 sunscreens approved
in the United States, there are 2 non-sun-
screen topical agents that are approved for the
treatment of conditions related to photo-in-
duced skin damage. These are tretinoin and
hydroquinone. Both are prescription prod-
ucts. Tretinoin is approved as an adjunctive
treatment for "mitigation of fine wrinkles,
mottled hyperpigmentation, and tactile
roughness of facial skin." Hydroquinone has
been marketed for decades for bleaching of hy-
perpigmented skin conditions.
The following section will only discuss sun-
screen agents.
8.1.2 Side Effects. Most common adverse
reaction to sunscreening agents are local. Sun-
screens can induce allergic, irritant, or photo-
toxic dermatitis. Sunscreens are frequently
blamed for allergic contact dermatitis and con-
tact urticaria. However, distinguishing sun-
screen-induced dermatitis from a solar-in-
duced condition can be difficult (229).
Moreover, the allergic reactions are, in many
cases, caused by fragrances, preservatives,
and other ingredients in the vehicle. Both ami-
nobenzoic acid derivatives and cinnamates are
known to cause skin sensitization. However,
aminobenzoic acid derivatives have lower sen-
sitization potential than the parent com-
pound, ABA (16). The latter was eliminated
461
European Union Australia
(16) Aminobenzoic acid
from most sunscreen formulations because of
frequent sensitization reactions. Photoaller-
gic reactions to benzophenones have been re-
ported (230). Some drugs including thiazide
diuretics, sulfonamides, and some local anes-
thetics such as benzocaine and lidocaine can
induce cross-sensitivity to aminobenzoic acid
derivatives. Physical sunscreens are ex-
tremely safe and non-sensitizing. The only re-
ported side effect to some physical sunscreens
is skin occlusion. Titanium dioxide has been
shown to be photochemically active in vitro,
but no such reaction has been shown in vivo
(231).
Several studies indicate that UVR irradia-
tion of organic sunscreens may induce
changes in the chemical structures of those
compounds (232). Some of the degradation
products have been shown to be capable of
damaging cell components including DNA
(233). This raises concerns over the photoirri-
tancy, photosensitization, and photomutage-
nicity potential of sunscreens, especially that
significant amounts of these photodegrada-
tion products may be absorbed through skin
(232). Reports on the carcinogenicity of sun-
screens are controversial. In vitro photogeno-
toxicity studies showed that, after UV irradi-
ation, sunscreens may attack the DNA to
produce mutation or cell death (233-236), pos-
sibly through the formation of free radicals
(234). However, there are no in vivo data to
support these conclusions. Furthermore, sev-
462
era1 acute and chronic in vivo studies demon-
strated that sunscreens protect from skin
damage and either prevent or delay carcino-
genesis (237). For a comprehensive reference
on sunscreens toxicity, the authors recom-
mend that the reader refers to Hayden et al.
(232)
8.1.3 Absorption and Disposition. Because
sunscreens are usually applied on a daily ba-
sis, and occasionally, over very large areas of
the body, there are concerns over the systemic
absorption and toxicity of these compounds.
Unfortunately, very little information is avail-
able on the pharmacokinetics and long-term
biological effects of sunscreens, although there
is evidence that sigrdicant systemic absorption
can occur (238, 239). Skin penetration of sun-
screening agents depends on several factors,
including the compound's molecular weight,
lipophilicity, and other physicochemical prop-
erties, as well as the composition and proper-
ties of the vehicle in which it is applied to the
skin (240,241).
There are few published data on the percu-
taneous absorption of sunscreens through hu-
man skin (239,242-248). Most of the informa-
tion came from in vitro penetration studies or
by estimation from the amount recovered in
the stratum corneum after tape stripping. The
rationale for using the latter method (often
referred to as the "reservoir technique") is
based on the finding by Treffel and Gabard
(248) that a linear relationship exists between
the drug concentration in the stratum cor-
neum and its in vivo percutaneous absorption.
Very few systemic absorption data have
been reported. Hayden et al. (238) reported
that 1-2% of a topical dose of benzophenone-3
(oxybenzone) (17) was excreted in the urine
(17) Oxybenzone (Benzophenone-3)
over a period of 48 h after a single application
of a sunscreen product (238). High subject
variability was reported in that study. Aranci-
Lifestyle and Over-the-counter Drugs
bia et al. (249) reported that 1.6-9.6% of p-
aminobenzoic acid (16) was recovered in the
urine of six volunteers after application of
three different formulations containing the
sunscreen. No significant difference was
found between the formulations. In an earlier
study, Feldman and Maibach (250) reported
that 28% of a topical dose of p-aminobenzoic
acid was recovered in the urine over 5 days
after application of the sunscreen as a solution
in acetone. It is possible the organic solvent
used in the latter study caused the consider-
ably higher systemic absorption.
I n vitro absorption studies (239, 251, 252)
showed small but significant penetration of
several organic sunscreens through the entire
thickness of human skin. For example, 0.03%
(251) and 0.4% (252) of octyl methoxycin-
namate (IS) were reported to be detected in
(18) Octyl methoxycinnamate
the receptor fluid in two different i n vitro pen-
etration studies. Less than 0.1% penetration
through the human skin was reported for ben-
zophenone-4 (19) (252). The data on benzo-
(19) Sulizobenzone (Benzophenone-4)
phenone-3 (oxybenzone) (17) varied from 1%
(251) to approximately 10% (239) of the ap-
plied dose. The higher percentages (5.8-10%)
were reported from a study that used several
commercial products.
Penetration and binding of organic sun-
screens into the stratum corneum has been
evaluated as a property that affects their re-
sistance to removal by water or perspiration.
The amount of sunscreen retained in the stra-
8 Sunscreens
tum corneum has been suggested to be di-
rectly related to the sun protection factor and
duration of action (248). Aminobenzoates
were reported to penetrate into the horny
layer of the stratum corneum and become
bound to the proteins in the epidermis by hy-
drogen bonding (253). This prolongs the dura-
tion of protection by aminobenzoates beyond
the time of their presence on the skin surface.
For organic sunscreens that do not penetrate
into the stratum corneum, the duration of ac-
tion depends on the length of their presence on
the skin surface. Highly lipophilic sunscreens
(e.g., octyl methoxycinnamate) have been
shown to have higher affinity to the stratum
corneum than water-soluble sunscreens (e.g.,
sulisobenzone) (252). However, penetration of
lipophilic sunscreens to deeper tissue and
hence their systemic absorption seems to be
limited (240). In an in vitro penetration study,
the percentage of applied dose of octyl me-
thoxycinnamate (18), sulisobenzone (191, and
octocrylene (20) that was retained in the stra-
(20) Octocrylene
tum corneum after 16-h exposure ranged from
4% for benzophenone-4 to approximately 10%
for octyl methoxycinnamate and octocrylene
(251). In the same study, 8.5% of the applied
dose of benzophenone-3 was retained in the
stratum corneum. In an in vivo penetration
study using skin-stripping method, 4% of ben-
zophenone-3 was found in the stratum cor-
neum after 30 min of application (254). The
formulation dependence of stratum corneum
penetration of three sunscreens [benzophe-
none-3 (17), octyl methoxycinnamate (181,
and octyl salicylate (2111 was demonstrated by
Treffel and Gabard (248,255).
(21) Octyl salicylate
The absorption of ZnO from intact skin af-
ter topical application is non-detectable. The
data on TiO, are controversial. Earlier studies
suggested that a very small amount of tita-
nium dioxide may penetrate the skin, but it is
unlikely that this would have any biological
significance (237). However, a recent in viuo
human study, in which skin punch biopsies
were collected after application of titanium di-
oxide, (256) showed that this sunscreen is
solely deposited on the outermost surface of
the stratum corneum and does not penetrate
into the deeper stratum corneum layers, the
epidermis or the dermis regardless of the sur-
face properties of the particles (256).
Very little is known about the distribution
and metabolism of sunscreens in humans, but
animal studies showed that benzophenone-3
undergoes substantial dermal metabolism and
protein binding in rats (257, 258), and is ex-
creted in both urine and feces. Excretion of
several sunscreening agents (including benzo-
phenone-3 and octyl methoxycinnamate) in
human breast milk after normal use has been
reported (259).
8.2 Physiology and Pharmacology
UV radiation has both acute and delayed ad-
verse effects on the human skin. The acute
effects are inflammation and sunburn, and the
delayed or chronic effects are primarily photo-
aging and photocarcinogenesis.
The susceptibility of individuals to the
harmful effects of UV radiation depends on
several factors: (1)skin pigmentation, (2)type
and amount of radiation, (3) skin hydration,
(4) thickness of the stratum corneum and epi-
dermis, and (5) the distribution and concen-
tration of peripheral blood vessels (260).

8.2.1 Acute Effects of UVR. The acute ef-
fects of UVR exposure are sunburderythema
and immediate pigment darkening (IPD).
Sunburn is a superficial skin burn with a mild
inflammatory reaction, the main manifesta-
tion of which is erythema. The symptoms may
also include tenderness, pain, and edema. It is
usually a first-degree burn, although occasion-
ally with extensive UVR exposure, a second-
degree burn that involves a thicker layer of
skin may develop. The main local symptom of
a second-degree burn is the development of
vesicles (blisters). In addition, systemic symp-
toms such as fever, weakness, and shock may
occur.
Erythema is the most common effect of
UVR exposure. The exact mechanism of how
WR induces skin erythema is not fully under-
stood; however, it is believed that a number of
mediators are involved in the inflammatory
reaction (260). These include histamine, lyso-
somal enzymes, kinins, and at least one pros-
taglandin. These mediators produce vasodila-
tation, which is manifested in erythema. As
the UV radiation penetrates into the epider-
mis, another inflammatory reaction involving
a lymphocytic infiltrate develops. Swelling of
the endothelium and leakage of red blood cells
also occurs. The inflammatory effect of UVB
radiation reaches its maximum in 12-24 h af-
ter exposure (260).
Microscopic changes in the skin in response
to UV radiation can be detected as early as 30
min after exposure (261). Epidermal changes
include intracellular edema, vacuolization,
and swelling of melanocytes and the develop-
ment of characteristic "sunburn cells." Sun-
burn cells, which were first described by
Daniels et al. (262),are photodamaged cells in
the process of undergoing apoptotic cell death
(224). They are dyskeratotic keratinocytes
with pyknotic nuclei and homogeneous eosin-
ophilic cytoplasm that develop in proportion
to the amount of UVB exposure. Sunburn cell
formation reaches a maximum at 18-24 h af-
ter exposure and resolves in 3-7 days.
Changes in the dermis include, initially, inter-
stitial edema and endothelial cell swelling, and
later, perivenular edema, degranulation and
loss of mast cells, decrease in the number of
Langerhans cells, neutrophil infiltration, and
erythrocyte extravasation. The dermal reac-
Lifestyle and Over-the-counter Drugs
tion peaks at 24 h after exposure and resolves
in 3-5 days. The later phase of microscopic
changes include hyperkeratosis, acanthosis
(epidermal thickening), disorganization and
misalignment of keratinocytes, dermal vascu-
lar ectasia, and mononuclear perivascular in-
filtration (261).
Erythema is primarily mediated by WB,
and its intensity is proportional to the dose of
UVB radiation. The reaction resolves in 3-5
days and initiates increased melanogenesis,
which reaches a maximum in 2- to 3-week
period.
Immediate pigment darkening (IPD) is an-
other acute effect of UVR exposure. IPD,
which is marked in some individuals and un-
detectable in others, is sometimes considered
as the initial phase of erythema (261). It is
mediated by UVA and lasts about 13-30 min.
The maximum wavelength for IPD is 340 nm
(263). IPD is thought to be a result of pho-
tooxidation process (224). The relative contri-
bution of UVA to sunburn and other acute ef-
fects of UVR is estimated at 18-20% (225).
8.2.2 Chronic Effects of UVR
8.2.2.1 Adaptive Responses. There are two
adaptive responses to the exposure to UV ra-
diation: thickening of the stratum corneum
and skin tanning (melanogenesis). Stratum
corneum thickening is mediated by m.,
whereas skin tanning can be induced by UVB
and UVA (225, 261).
Thickening of the stratum corneum occurs
as an adaptive response to prolonged UVB ex-
posure. The stratum corneum proliferates
through increased synthesis of keratin by
basal keratinocytes.
Skin tanning, or delayed pigment darken-
ing, results from the production of melanin
and serves as a protective mechanism against
further UVR-induced damage. Skin tanning is
mainly induced by UVB, with a wavelength
spectrum similar to that of erythema (224).
UVB induces melanogenesis by enhancing the
binding of circulating melanocyte-stimulating
hormone (MSH) to melanocytes. This leads to
melanocyte proliferation, dendritic arboriza-
tion, and melanin production. The melanin is
then distributed to the surrounded keratino-
cytes (261). Melanin absorbs, reflects and scat-
ters UV light and is a free radical trap. UVA
8 Sunscreens
induces a delayed tanning reaction. This mel-
anogenesis reaction involves oxidation of pre-
existing melanin followed by new and in-
creased synthesis of melanin. It is also
accompanied by increased population density
of melanocytes with increased production of
melanosomes and increased melanization of
the epidermis (225).
8.2.2.2 Photoaging. Premature photoag-
ing is one of the long-term effects of exposure
to sunlight (264). It is induced by prolonged
exposure to all portions of the solar spectrum
including W A , UVB, and infrared (261). The
incidence and severity of photoaging is be-
lieved to be proportional to the cumulative
dose of WR (237). There is evidence that
chronic exposure to suberythemal doses of
UVA produces changes in human skin indica-
tive of photoaging (265-267).
The common manifestations of skin photo-
aging are dryness, roughness, irregular pig-
mentation, actinic keratosis, wrinkling, elas-
tosis, inelasticity, and sebaceous hyperplasia
(264). Although the physical appearance of
photoaging is similar to normal aging, there
are histological and biochemical differences
that distinguish each condition from the other
(261). In addition, photoaging can be slowed
down and even reversed by reduction of UVR
exposure (including using sunscreens), or by
other treatments such as retinoic acid,
whereas normal aging is irreversible (268).
The typical macroscopic appearance of photo-
aged skin results mainly from break down of
the skin elastic fibers (elastosis) caused by
long-term exposure to UV light. The exact
mechanism is not fully understood. Other
changes associated with photoaging include
cracking, telangiectasia (spider veins), ecchy-
moses (subcutaneous hemorrhagic lesions),
and hyperpigmentation. Solar radiation also
decreases the regenerative capacity of the skin
fibroblasts and keratocytes and therefore ac-
celerates skin aging (261).
8.2.2.3 Carcinogenesis. Skin cancer is the
most serious adverse effect of UVR. There are
three common types of skin cancer: mela-
noma, basal cell carcinoma (BSC), and squa-
mous cell carcinoma (SCC). BSC and SCC are
known collectively as non-melanoma skin can-
cer (NMSC). BSC is the most common skin
cancer, but it has the lowest mortality rate
(269). Malignant melanoma is the most seri-
ous type of skin cancer.
Although the link between skin cancer and
exposure to sunlight is very strong, the sus-
ceptibility of individuals to the carcinogenic
effect of UV radiation varies depending on sev-
eral factors including skin pigmentation, age,
sex, and phenotype. Particularly, factors such
as fair skin, blue eyes, red or fair hair, and
inability to tan have been linked to increased
risk of NMSC in several studies (270-276).
Both chronic human studies and animal
studies proved the causal relationship be-
tween cumulative UVR exposure and skin
cancer (2771, particularly non-melanoma skin
cancer (NMSC). The link between the two is
also evident from the fact that NMSC is most
common on the head, neck, arms, and hands
(278, 279). In particular, the correlation be-
tween UVR exposure and SCC seems to be
very strong. Cutaneous SCC of the head and
neck occurs almost exclusively on areas receiv-
ing maximal exposure (269,280). The link be-
tween BCC and cumulative exposure to UVR
is not as evident (270, 281). Although BCC
occurs on the face, head, and neck, unlike
SCC, its distribution does not correspond well
with the areas that receive maximum sun ex-
posure (269). Case-control studies indicated .
that cumulative sun exposure is the most im-
portant risk factor for SCC, whereas inability
to tan was the most important risk factor for
BCC (270,282,283).Subsequently, it has been
suggested that, for BSC, intermittent sun ex-
posure, particularly in the childhood, may be
more important than cumulative exposure
(282).
The data on a causal relationship between
cumulative W R exposure and malignant mel-
anoma is not conclusive, but there is strong
evidence that intermittent intense sunlight
exposure, particularly severe sunburn in
childhood, is a major risk factor for develop-
ment of malignant melanoma (284-286).
There is scientific evidence that UVB can in-
duce melanocyte proliferation in both exposed
and covered areas of the body (2871, which ex-
plains why malignant melanoma sometimes
occurs in unexposed parts of the body.
Both W A and UVB can induce DNA dam-
age, and photocarcinogenesis has been re-

ported following repeated UVA exposure in ro-
dents (288). In addition, UVA is believed to
augment the development of UVB-induced
non-melanoma skin cancer (287).
The first step in UVR-induced skin cancer
is UVR-initiated DNA mutation, which causes
the transformation of the normal cells to ma-
lignant cells. For UVR to initiate a biological
reaction, it has to be absorbed by endogenous
molecules (chromophores). UVB is absorbed
directly by the DNA, and therefore can di-
rectly induce DNA mutation (2241, in the form
of thymine dimer formation (289). Some pro-
tein components may also a d as chro-
mophores for UVB (224). W A is absorbed by
the reduced forms of the co-enzymes nicotin-
amide adenine dinucleotide (NADH) and nic-
otinamide adenine dinucleotide phosphate
(NADPH), tryptophan, riboflavin, and mela-
nin (224, 290). UVA-induced DNA damage is
believed to be mediated by oxygen reactive
species that are released after the absorption
of UVA by those endogenous chromophores
and results in photooxidation of selected bases
(224,290,291). The induced damage may take
the form of single-strand breaks, induction of
thymine dimers, or DNA-protein crosslinks
(290,291). In addition, UVA I1may be directly
absorbed by the DNA similar to UVB (224).
It is believed that UVR exposure causes
skin immune suppression. Both UVB and
UVA have been implicated (292). A possible
mechanism is UVA-induced lipid peroxida-
tion, which results in the migration of im-
mune-mediating cells from the epidermis and
therefore leads to skin immune suppression
(224). It has been suggested that skin immune
suppression plays a role in carcinogenesis.
However, the relationship between the two is
not well understood yet (293).
8.2.3 Mechanisms of Action of Sunscreens.
Sunscreens delay the induction of sunburn by
absorbing or reflecting a portion of the UVR
reaching the epidermis. Organic sunscreens
are aromatic compounds that absorb light en-
ergy in the UV region, and therefore reduce
the amount of UVR reaching the stratum cor-
neum. A benzene ring has the ability to trans-
form high energy UVR into harmless long
wave radiation above the 380-nm range, which
is emitted from the skin as heat (294). The
Lifestyle and Over-the-counter Drugs
conversion of UVR to longer wavelength oc-
curs through resonance delocalization. Most
chemical sunscreens absorb up to 95% of the
UVB spectrum, but do not absorb in the UVA
range. Avobenzone (22) is the only organic
(22) Avobenzone
sunscreen that provides a high degree of UVA
protection. Benzophenones and anthranilates
provide partial protection in the UVA range.
Sunscreens are almost always used in combi-
nations to broaden the absorbance spectrum
and increase the SPF value of the product.
Little is known regarding the quantitative
ability of sunscreens to prevent UVR-induced
adverse effects other than sunburn. For exam-
ple, there is no information on the threshold
or dose-response for UVR-induced immuno-
suppression and DNA damage (2371, but there
is strong evidence that regular use of Sun-
screens reduces the incidence of precancerous
lesions. A large population study in Australia
proved the effectiveness of sunscreens in re-
ducing the incidence of solar keratosis (291).
Solar keratosis is a known precursor for squa-
mous cell carcinoma and an established risk
factor for basal cell carcinoma and melanoma.
It has been suggested that sunscreens aug-
ment the defense mechanism against oxida-
tive damage by UV-generated free radicals.
This defense mechanism is mediated by thio-
redoxin reductase in the human keratocytes,
which reduces superoxide anion radicals
through hydrogen peroxide to water (295).
However, the oxygen radicals concentrations
generated by UVA and UVB radiations, even
below the minimal erythemal dose, are high
enough to cause considerable deactivation of
thioredoxin reductase. Sunscreens have been
shown to protect the thioredoxin reductase
against both UVA and UVB in human skin of
types I and 11. The same sunscreen, however,
8 Sunscreens
failed to protect the enzyme in more pig-
mented skin (types I11 and IV).
8.2.4 Sun Protection Factor. Sunscreens
were originally developed to prevent sunburn
and minimize erythema (224). The efficacy of
sunscreen products is rated by their sun pro-
tection factor (SPF), which is defined as the
ratio of W energy required to produce a min-
imal erythemal dose (MED) on protected skin
to the W energy required to produce MED on
unprotected skin. It is calculated as the ratio
of time of W exposure necessary to produce
minimal erythema in sunscreen protected
skin to that time in unprotected skin. This
SPF measures the effect of UVB only and does
not account for the W A effect. Few methods
have been proposed for a W A protection fac-
tor, including an in vivo pigment darkening
method, and an in vitro critical wavelength
test proposed to the FDA by the Cosmetic, Toi-
letry and Fragrance Association (CTFA).
However, there is still no generally acceptable
method for UVA testing, and the FDA has not
yet reached a conclusion. It is also noteworthy
that the SPF is not a measure of other photo-
damage protective properties of sunscreens
(e.g., protection against skin cancer and im-
munologic protection). However, it is gener-
ally agreed that a higher SPF sunscreen will
provide better protection against UVB-in-
duced damage. There have been several at-
tempts at determining the immune protection
factor of sunscreens (293,296). All these stud-
ies seemed to indicate some immune protec-
tion of sunscreens, but with no correlation
with the SPF. It seems that the extent of im-
mune protection is much less than erythema
protection, which suggests that the wave-
lengths that cause immune suppression are
different from those that cause erythema
(293).
8.3 Chemistry and Structure-Activity
Relationships
The chemical sunscreens approved for OTC
use in the United States are classified into
seven classes: aminobenzoates (previously
known as PABA and PABA derivatives), sa-
licylates, cinnamates, benzophenones, an-
thranilates, dibenzoylmethane derivatives,
and one miscellaneous chemical (phenyl-ben-
zimidazole sulfonic acid, 23). I n addition,
there are several camphor derivatives that are
marketed as sunscreens in Europe, but none
(23) Phenylbenzimidazole Sulfonic Acid
are approved in the United States. The mini-
mal structural requirements for absorbance in
the UVB and W A regions are an aromatic
ring with an electron-releasing group and an
electron-accepting group either in the ortho-
or para-position from each other. This struc-
tural arrangement facilitates resonance delo-
calization. The more easily the compound res-
onates, the lower the required quantum
energy for the electron transition, and hence
the longer the maximum absorbance wave-
length, because A is inversely proportional to
the energy. In addition, the spectral properties
of sunscreens may be affected by dielectric ef-
fects, solvent-solute interactions, and pH of
the vehicle (232). An excellent analysis of the
structure-activity relationships of sunscreen ,
chemicals has been reported by Shaath (297).
The following discussion is largely based on
his findings.
8.3.1 Aminobenzoates. In aminobenzoic
acid (161,the parent compound of this class,
the presence of the amino group and carboxy-
lic acid group in the para-position from one
another allows for the electron transition re-
sponsible for absorption in the UVB region.
However, the very position of the two groups
results in a number of properties that are un-
desirable for a sunscreening agent. These in-
clude the following (297):
0 Susceptibility of the free amine to oxidation
Intermolecular hydrogen bonding resulting
in a crystalline physical state: crystalline
sunscreens are difficult to incorporate in
topical products and may result in an unac-
ceptable product if the sunscreen agent is
not solubilized properly

0 High aqueous solubility because of excessive
hydrogen bonding with the solvent and
therefore shorter retention time on the skin,
especially with perspiration
0 A dramatic solvent effect, which shifts the
A,, from 293 nm in nonpolar solvents to
266 nm in polar solvents: this influences the
efficacy of sunscreen products
Because of the structural limitations of
ABA, several sunscreening chemicals based on
ABA structure were developed with attempts
to capitalize on the strengths and eliminate
the drawbacks. There are four compounds
that successfully addressed the problems of
ABA. These are N,N-dimethyl PABA ethyl es-
ter, N,N-dimethyl PABA butyl ester, N,N-di-
methyl PABA amyl ester (padimate A), and
N,N-dimethyl PABA octyl ester (padimate 0).
Padimate 0 (24) has the most desirable prop-
(24) Padimate 0
erties in this class. The molar extinction coef-
ficient of padimate 0 is 28,400 in polar sol-
vents, more than twice that of ABA, and one of
the highest values among all chemical sun-
screens. The solvent effect shift is much
smaller than that ofABA (from 300 nm in non-
polar solvents to 316 nm in polar solvents).
Moreover, eliminating the intermolecular hy-
drogen bonding, and the subsequent decrease
in intermolecular association, led to the for-
mation of a liquid instead of crystalline solid,
which is a major advantage for formulating
sunscreen products (297).
8.3.2 Salicylates. Salicylates are ortho-hy-
droxybenzoic acid esters. The presence of the
hydroxyl group in the ortho- position allows
Lifestyle and Over-the-counter Drugs
for intramolecular hydrogen bonding, which
lowers the energy requirements for the com-
pound to be prompted to the excited state.
This results in a UV absorbance in the 300-nm
range, compared with 270 nm for the para-
hydroxybenzoates (parabens). Furthermore,
the ortho-disubstitution seems to stabilize
these agents against solvent effect. This is also
attributed to intermolecular hydrogen bond-
ing (298). On the negative side, the ortho-sub-
stitution causes crowding and strain on the
molecule, which results in deviation from pla-
narity and hence a low extinction coefficient.
Although salicylates are weak UV absorbers,
they are very popular sunscreens because of
their excellent safety record and their physico-
chemical properties that are favorable for cos-
metic formulations. They are water-insoluble
liquids and excellent solubilizers for other in-
soluble cosmetic ingredients and sunscreens
such as benzophenones. 2-Ethylhexyl salicy-
late (21) (also known as octyl salicylate) is the
most commonly used sunscreening salicylate.
The 2-ethylhexyl moiety is a common sub-
stituent in other popular sunscreens (2-ethyl-
hexyl p-methoxycinnamate, 2-ethylhexyl di-
methyl p-aminobenzoate, and 2-ethylhexyl-2-
cyano-3,3-diphenyl acrylate). The moiety
ensures the compounds' insolubility in water,
and makes them useful for water-proof sun-
screen formulations (297). Other salicyfates
that are approved for OTC marketing in the
United States are homosalate (25) and trieth-
anolamine salicylate (26).
(25) Homosalate
(26) TEA Salicylate
8 Sunscreens
8.3.3 Cinnamates. Cinnamates have an
unsaturated bond conjugated both to the aro-
matic ring and to the carbonyl group, which
allows the electron delocalization to occur
throughout the molecule. This results in a UV
absorbance at about 305 nm and high molecu-
lar extinction coefficient, which makes this
group strong UV filters. One of the best
designed cinnamates is 2-ethylhexyl p-meth-
oxycinnamate (octyl-p-methoxycinnamate).It
has an electron releasing methoxy group in
the para- position, which further facilitates
the electron delocalization process. The 2-eth-
ylhexyl group makes it practically insoluble in
water. In addition to octyl-p-methoxy cin-
namate, cinnoxate (27) is also included in the
(27) Cinoxate
sunscreens monograph. One of the drawbacks
of cinnamates is their moderate photostability
caused by cis-trans isomerism (297).
8.3.4 Benzophenones. Benzophenones are
aromatic ketones. In this group, the electron
donating substituent is located in the para-
position to the carbonyl group. The carbonyl
group acts as the electron-acceptinggroup and
participates in the resonance delocalization
process. Aromatic ketones resonate easier
than esters and therefore absorb at a longer
wavelength (A, > 320 nm), which puts them
slightly into the UVA region. The main prob-
lems with benzophenones are as follows (297):
They are crystalline solids that are difficult
to solubilize in cosmetic formulations
Some benzophenones are susceptible to
large shifts in their UV maxima because of
solvent effect
Unlike esters, which are rapidly hydrolyzed
in the body, benzophenones maybe poten-
tially toxic if absorbed through the skin: sta-
tistically, more frequent allergic reactions
have been reported with oxybenzone than
with PABA (299). Three benzophenones are
listed in the sunscreens monograph: dioxy-
benzone (28), oxybenzone (17), and suliso-
benzone (19).
(28) Dioxybenzone (Benzophenone-8)
8.3.5 Anthranilates. Anthranilates are or-
tho-aminobenzoates. There is only one an-
thranilate compound approved as a sun-
screening agent in the United States, and two
anthranilates in Europe. The ortho- position-
ing of the amino group results in intramolec-
ular hydrogen bonding and greatly enhances
the resonance delocalization. The result is a
A,, of 336 nm for menthylanthranilate com-
pared with 298 nm for PABA (the para- sub-
stituted amino benzoate). Similar to the case
with salicylates, the molar extinction coeffi-
cient of anthranilates is much lower than that
of aminobenzoates because of the steric
crowding that causes the compound to deviate
from planarity. Also, similar to salicylates, an-
thranilates are stable and do not exhibit sig-
nificant solvent effect (297,232). .
(29) Menthyl anthranilate
8.3.6 Dibenzoylmethanes. Avobenzone (22)
is the only member of this class that is ap-
proved for use in the United States There are
two other dibenzoylmethanes that are ap-
proved in Europe in addition to avobenzone.
Dibenzoylmethanes are substituted dike-
tones, with a W spectrum well into the W A
region. This unique spectrum among W fil-
ters is a result of the keto-en01tautomerism.
The W A absorbance of these compounds is

mainly caused by the en01 form, which has a
A,, greater than 345 nm, whereas the keto
form exhibits a A,, of about 260 nm. The
presence of the hydroxyl group in an ortho-
position to the carbonyl group shifts the equi-
librium of the keto-en01 tautomerism toward
the en01 form and away from the keto. Diben-
zoylmethanes have a high molar extinction co-
efficient, but their main drawback is their low
photostability and susceptibility to photoi-
-
somerization. which results in an irreversible
loss of activity. The keto form is more suscep-
tible to photoisomerization than the en01
(297). Dibenzoylmethanes are relatively sta-
ble in polar solvents (alcohols),but unstable in
nonpolar solvents (300).
8.3.7 Physical (Particulate) Sunscreens. Zinc
oxide and titanium dioxide are the two metal
oxides approved as sunscreens in the United
States. Microfine particles of these metal ox-
ides, with a mean particle size of 0.2 pm or
less, and a narrow particle size distribution,
are used in physical sunscreen products. The
average particle size is below the optimal light
scattering size, which allows visible light to be
transmitted and therefore renders the prod-
uct virtually invisible on the skin (237, 290).
Zinc oxide is primarily a W absorber, whereas
titanium dioxide is predominantly a reflector,
although it does absorb UV light both in the
UVB and UVA region. Zinc oxide strongly ab-
sorbs UVR from about 380 nm all the way
through the UVB and into the UVC region,
and therefore, was commonly referred to as
UV block. Its UVA protection is superior to
that of titanium dioxide, mainly because of the
stronger absorbance. The efficacy of particu-
late sunscreens depends largely on the refrac-
tive index, particle size, particle morphology,
dispersion in the vehicle, and film thickness
(231). Microparticulate sunscreens are fre-
quently coated with silicones andlor alumina
to aid their dispersibility.
8.4 History
8.4.1 Emergence of Sunscreens. The first
commercial sunscreen product appeared in
the United States in 1928. It was an emulsion
of benzyl salicylate and benzyl cinnamate. In
the early 1930s, a 10% solution of phenyl sa-
Lifestyle and Over-the-counter Drugs
licylate was developed and marketed in Aus-
tralia. In 1935, quinine oleate and quinine
bisulfate were marketed in the United States.
p-Aminobenzoic acid (PABA) was patented in
1943, and was for many years the leading or-
ganic sunscreen. Several aminobenzoates
(PABA derivatives) were introduced subse-
quently. PABA derivatives remained the pri-
mary sunscreen ingredients during the 1960s
and 1970s. The first reference to particulate
sunscreens appeared in 1947 (301) in an arti-
cle titled "Zinc Oxide in Face Powders." The
article discussed the absorption spectra of
metal oxides, including zinc oxide and tita-
nium dioxide. In the same article, the author
predicted the value of zinc oxide as a broad
spectrum UV block, based on its absorption
spectrum. The first report on titanium dioxide
as a sunscreening agent appeared in 1952
(302). However, the first commercial use of
microparticulate metal oxides as sunscreens
was in 1989 with the introduction of a tita-
nium dioxide product. Zinc oxide was intro-
duced in 1991 (303).
The US military contributed to the intro-
duction of new sunscreens as well as increas-
ing the public awareness and widespread use
of sunscreening agents. The first reported use
of red petrolatum was by the U.S. military
during World War I1 (304). They also ysed
other agents such as glycerol-PABA and sev-
eral salicylate derivatives. Moreover, the U.S.
military issued the first specifications on sun-
screens in 1951, in which it listed approved
sunscreens and their recommended concen-
trations (297). However, most of the sun-
screen compounds and sunscreen research
originated from the cosmetic and personal
care industry. The SPF testing, as a means of
quantifying the efficacy of sunscreens, started
in the 1960s,and was initially known as Light
Protection Factor (305).
8.4.2 History of FDA Regulations. Although
the FDA did not start regulating sunscreens as
OTC drugs until 1978, it has in fact, consid-
ered them drugs as early as 1940. This posi-
tion concerning the legal classification of sun-
screens came in a trade correspondence to the
cosmetic industry (306), in which the FDA
stated: "Articles which refer to sunburn or
any other disease condition are drugs under
8 Sunscreens
Section 201(g), but articles which are repre-
sented exclusively for the production of an
even tan will be regarded as cosmetics under
Section 201(i)." In 1976, in another correspon-
dence to the cosmetic industry, the FDA
stated "We have concluded that a product con-
taining a sunscreen ingredient . . . even when
labeled solely as a tanning aid, is both in-
tended and understood to be a preventative of
sunburn, and is therefore a drug" (307). On
August 25, 1978, the FDA published the first
tentative monograph on sunscreens (308).
The monograph contained 21 W filters and
their acceptable usage levels. However, the
FDA did little to regulate sunscreen-contain-
ing products manufactured by the cosmetic
industry until the mid-1980s. Until then, cos-
metic products containing sunscreens contin-
ued to be marketed as cosmetics, unregulated,
as long as they were correctly labeled as cos-
metics and did not make clear therapeutic
claims. In 1985, the FDA reiterated its previ-
ous position on sunscreen classification in a
memorandum of meeting with the cosmetic
industry (307), and in 1987, it launched a ma-
jor regulatory campaign against cosmetic
products with antiaging claims, many of
which contained sunscreens. In warning let-
ters to the marketers of these products, the
FDA cited them as drugs under Section 201(g)
of the Food and Drug Act. Moreover, those
products that contained sunscreens unap-
proved by the FDA were cited as "new" drugs
within the meaning of Section 201(p) (307).
In May 1993, the FDA issued the tentative
final monograph for OTC drug sunscreen
products (309), which included 20 sunscreen
actives. In the monograph, the FDA redefined
cosmetic products that contains any sun-
screen, use the term sunscreen, claim an SPF,
or make any therapeutic claim about sun pro-
tection as drugs. Since then, the FDA has
made it clear that any sunscreen agent not
eligible under the OTC Drug Review process
must go through an NDA to be approved for
use in the United States. In 1994, the FDA
published a proposed amendment to the ten-
tative monograph (310) announcing its inten-
tion to delete five sunscreens for lack of inter-
est from the industry. The agency proposed to
keep only the 15 actives for which USP mono-
graphs existed or were being developed. The
deleted agents were hardly used in any sun-
screen product in the United States.
During the late 1980s and early 1990s,sun-
screen products started moving away from be-
ing purely beach and recreational products to
being part of daily skin care. This occurred
largely as a result of a public education cam-
paign that started in the 1980s because of the
increased scientific evidence on the correla-
tion between skin cancer and long-term expo-
sure to UVR.In 1989, UVA protection started
to become an issue in the United States. Until
then, the tentative monograph contained no
broad-spectrum UVA filters and no mention of
UVA. In early 1993, Roche petitioned to the
FDA to include avobenzone, a broad spectrum
UVA filter that it developed in 1971 and had
been widely used in Europe since 1981. How-
ever, when the FDA issued the tentative final
monograph for OTC sunscreens in May 1993,
avobenzone was not included. In the following
years, the FDA came under pressure from
Roche, the CTFA, and the American Academy
of Dermatology to recognize avobenzone as a
safe and effective (Category I) sunscreen. In
September 1997, the agency amended the ten-
tative final monograph to include avobenzone
as a Category I OTC sunscreen (311). Before
this amendment, the FDA had approved a sin- ,
gle product containing avobenzone (Shade
UVA Guard from Schering-Plough). The ap-
proval of Schering-Plough's product came af-
ter an NDA. Zinc oxide, which was until then
considered Category I11 (available data are in-
sufficient to classify as safe and effective and
further testing is required), was also added to
the tentative final monograph in October 1998
(312).
On May 21,1999, the FDA issued the final
monograph of OTC sunscreen products (313).
The final monograph contained the 16 agents
listed in Table 9.11. Those are the agents that
currently have USP monographs. The FDA
kept two other agents under consideration in
case monographs are developed in the future.
The two agents are diethanolamine methoxy-
cinnamate (30) and lawsone (31) with dihy-
droxyacetone. The final monograph also con-
tained new regulations for SPF testing and
labeling. The FDA gave sunscreen makers 2
years to comply with the new regulations. The

(30) DEA Methoxycinnamate
(31) Lawsone
effective date was later extended to December
31, 2002, in response to a petition submitted
by the CTFA (314).
8.5 Current and Future Trends
It is evident from the number of new cases of
skin cancer that there is a need for new sun-
screens with better protection. However, the
cost and time required for introducing new
sunscreen agents through an NDA is prohibi-
tive. Therefore, a large portion of the current
research in the field focuses on novel drug de-
livery systems to prolong the duration of the
sunscreening effect, enhance the water resis-
tance (substantivity) of the products, enhance
their SPF, or decrease their irritation poten-
tial. Another important area is improving the
aesthetics of sunscreen products, particularly
physical sunscreens.
Several new sunscreening compounds have
been introduced in Europe, Australia, and Ja-
pan in the past few years. Two of the most
interesting new agents are bis-ethylhexyloxy-
phenol methoxyphenol triazine (BEMT) and
methylene bis-benzotriazolyl tetramethylbu-
tylphenol (MBBT). BEMT is a potent broad-
spectrum UV-absorber that covers the full
UVA and UVB spectrum. In contrast to avo-
benzone (the only broad spectrum UVA filter
available in the United States), BEMT has a
good photostability (315). MMBT (316) repre-
sents a new class of broad-spectrum UV filter
(organic microparticulates). The product con-
Lifestyle and Over-the-counter Drugs
sists of microfine organic particles that are dis-
persed in the aqueous phase of sunscreen
emulsions (315, 316). It is equally protective
against UVA and UVB radiation (UVA/UVB
protection ratio = 1.0), and is extremely pho-
tostable. The safety and lack of genotoxicity of
these two agents have been demonstrated
both in vitro and in vivo. The European
Unions Scientific Committee on Cosmetic
Products and Non-Food Products Intended
for Consumers (SCCNFP) has approved the
two compounds at concentrations up to 10%.
In September 2000, Ciba Specialty Chemicals
has petitioned with the FDA to add BEMT and
MMBT to the OTC sunscreen monograph
(315).
Alternative approaches for photodamage
repair and protection is an emerging trend in
sun protection. Antioxidants (particularly vi-
tamin E) are common ingredients in commer-
cially available sunscreens. It has been previ-
ously shown that topical application of
vitamin E inhibits UVR-induced cellular dam-
age, edema, and erythema (317). In the past
few years, there has been rising interest in
scientific research to prove the efficacy of an-
tioxidants as chemopreventive agents (318,
319). Recent work demonstrated evidence of
DNA photoprotection of a-tocopherol (a vita-
min E compound) (317). In 2001, an oral anti-
oxidant (astaxanthin) was promoted as sun
protectant pill (320). The FDA approved the
marketing of astaxanthin as dietary supple-
ment in 1999, but the product did not undergo
the review for sun protection claims. Astaxan-
thin is a carotenoid obtained from micro algae
and is over 500 times more potent antioxidant
than vitamin E. Other natural products that
gained considerable attention as potential
chemopreventive agents are tea polyphenols,
resveratrol (a natural product from grapes),
ginger compounds, curcumin, diallyl sulfide (a
compound from garlic), and several plant fla-
vonoids, such as apigenin, catechin, silymarin,
and genistein (318, 319, 321, 322). A number
of these compounds are found in skin care
products, with no cancer prevention claims.
Chemoprevention has a large potential to
become a viable approach for skin cancer pre-
vention. However, well-designed case-control
clinical studies are needed to prove its efficacy
(321).
9 Future Trends for OTC and Lifestyle Drugs
With regard to FDA regulations, there re-
main few areas of concern. The main ones are:
the determination of a standardized UVA test-
ing methodology (with in vivo relevance), and
the testing and labeling of sunscreen drug
products with SPF values above 30. The FDA
is concerned that high SPF number may en-
courage consumers to extend their exposure to
the sun (314).
9 FUTURE TRENDS FOR O T C A N D
LIFESTYLE DRUGS
What will the future of OTC look like? Back in
the 1990s, Rx-to-OTC switches were deemed
to be the driving force for growth in the OTC
industry. We did see rapid growth in the mid-
1990s, but the number of switches has dwin-
dled substantially near the end of the 20th
century (323). The so-called "easy ones" have
been switched. Pharmaceutical companies
need to be creative with future switches. Sev-
eral seemingly promising switches such as the
statins and acyclovir have been denied by the
Advisory committee (35). Refusal by the FDA
to switch a product in the past does not mean
there is no opportunity for future switch. It
simply means the sponsor will need to develop
more data. Most switches need to go through a
few passes in front of the Advisory Committee
before they are eventually switched. Some of
the therapeutic categories such as hair growth
and smoking cessation that have not been in-
corporated into the monograph system by the
OTC review panel were eventually switched.
The reason why these OTC products existed
before the OTC review strongly suggests that
those are unmet consumer needs. They got
switched when the right actives came along
and the right studies were done to convince
the FDA that self-medications of these prod-
ucts are possible (33). The timing and regula-
tory environment has to be right. By the same
analogy, some of the previously denied Rx-to-
OTC switch candidates may be resurrected as
more safety data are accumulated. The fact
that healthcare providers would like to control
cost will lead to more citizen petitions as in the
case of non-sedative antihistamines. When
these candidates will be switched will depend
on how the politics play out. The innovator
companies said that the FDA has no authority
to force them to switch. However, the same
picture may not hold in a country like the U.K.
The MCA has been aggressively promoting
switches.in recent years, and they want to ac-
celerate them in the next few years to control
costs (48).
No matter what the future of Rx-to-OTC
switch will be, one thing for sure is that future
switches will be driven by science and an evi-
dence-based medicine approach as in the past.
Even though most of the OTC products are for
acute use, there are already a few products
such as hair growth products and smoking ces-
sation product for chronic use. The desire of
the baby boomers to maintain their vigor and
the desire of healthcare providers to control
cost favors preventive OTC products, which
will continue to challenge the chronic use bar-
rier. The switch of these kinds of products will
be enhanced by the advance of diagnostic
products (324).
The future of the OTC industrv " will also be
different because of dietary supplements and
cosmeceuticals. The boundary between them
and OTC products will be blurred (325). The
FDA has already issued draft guidance for reg-
istering herbal drug products as either OTC or
prescription products (326). In countries such
as Germany and Japan, herbal and dietary
,
supplements have been registered as drug
products for many years. The current lax reg-
ulatory environment may not favor the extra
efforts of registering an herbal product as a
drug product. However, it may become a wor-
thy attempt as the quality standard demanded
on the dietary supplement industry becomes
tighter. Here again, the science will dictate the
success of the conversion. Similar changes
may occur with cosmeceuticals. The FDA will
undoubtedly work out a proper procedure for
how to regulate the so-called cosmeceuticals.
Some of them may first become Rx before they
will be switched to OTC.
For a long time, the FDA has not required
any adverse event reporting for OTC mono-
graph products. However, this will be the fo-
cus of the agency and the pharmaceutical com-
panies to place a high priority on adverse
event reporting. This not only represents re-
sponsible marketing but also can be used to
enhance public relationships.
474
The recent merger of pharmaceutical com-
panies and the demand for double-digit
growth have put the OTC divisions of the con-
solidated companies in a very demanding po-
sition. What benefits besides growth does the
OTC division bring to the table? Perhaps one
important function the OTC division serves is
for lifestyle management through Rx-to-OTC
switch. The other benefit is their direct-to-
consumer advertising expertise. The prescrip-
tion businesses are rapidly catching up in this
area.
The advance of communication technology
will also significantly alter the landscape of
the OTC industries. There are already compa-
nies that provide data-logging devices that al-
low transfer of diagnostic data between pa-
tients at home and the physician's office (327).
Right now, the technology is limited to immo-
bile patients. The advance of handheld wire-
less devices and cellular hones will make
transfer of medical information much easier
for mobile OTC consumers. With the advance
of better internet security, personal data may
be readily portable (328). It is not hard to im-
age that consumers can cany a credit card-like
device that has all hisher medical history and
possible drug-drug interactions stored on the
device.
Pharmaceutical companies and govern-
ment used to the two major drivers for Rx-to-
OTC switches and they will continue to be the
maior
" drivers for the switches. The former
conducts switches for lifestyle management of
prescription drugs while the latter promotes
switches to reduce national prescription costs.
In future, manage care providers will also be a
major driver to petition for switches as in the
case of second-generation antihistamines.
The growth in the OTC industry will come
not only from additional switches but also
from a lot of "think outside the box" ap-
proaches. One thing for sure, the industry can-
not keep marketing additional flavors to grow
their business. The business model will have
to change!
What is the future for lifestyle drugs? The
future is very promising despite the strong re-
sistance of the healthcare providers to pay for
lifestyle drugs simply because the consumers
want them to feel and look better. With the
advances in our understanding of causes of
Lifestyle and Over-the-counter Drugs
diseases and disorder and genomic research,
pharmaceutical scientists can now create
drugs to target receptors that were previously
unknown. To maintain double-digit growth,
the pharmaceutical industry cannot just focus
on unmet consumer needs in the area of life-
threatening conditions such as cancer,
asthma, anti-infective diseases, and cardiovas-
cular diseases. They also need to capitalize on
unmet consumer needs in the area of lifestyle
enhancement to the aging consumers. The
growth in the market for lifestyle drugs will
likely be faster than that for traditional life-
threatening diseases. The allowance of direct-
to-consumer advertising creates a favorable
environment to market lifestyle prescription
drugs to informed consumers. The advances in
genomic research will theoretically allow for
more precise targeting, thereby minimizing
unwanted side effects. This will facilitate the
acceptance of lifestyle drugs and possible
switch to OTC because of their lower side ef-
fects. Several lifestyle products such as hair
growth and smoking cessation have already
been switched and became quite successful.
The issue of quality of life is as important for
treating life-threatening diseases especially in
developing countries. Our stressful lifestyle
will in turn cause many more lifestyle diseases
such as mild depression, anxiety, ulcer, and
other psychosomatic diseases. In fact, the op-
portunities for lifestyle drugs in treating psy-
chosomatic diseases are endless considering
the extensive classifications in the Diagnostic
and Statistical Manual of Mental Disorders
(329).
Pharmaceutical companies will need to in-
crease the awareness of the consumers about
their lifestyle products, but they need to be
very careful in advertising them. They need to
cultivate a positive image of responsible mar-
keting. Roche is already doing that in adver-
tising their product Xenical, which is targeted
for a special group of diabetic patients that has
a medical need to lose weight. The company
will also need to tailor their advertising to cre-
ate psychological ties with the patients and
ensure compliance. All lifestyle products have
dedicated websites to educate and provide
product information to consumers. Careful
References
consumer marketing will be needed to identify
how to best position and advertise the prod-
ucts.
The lifestyle drugs are here to stay and
their definition will continue to change. Their
market will continue to grow as the baby
boomers age.
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CHAPTER TEN

Radiopaques
C. T. PENG
University of California
Department of Pharmaceutical Chemistry
School of Pharmacy
San Francisco, California
and
San Jose State University
Nuclear Science Facility
San Jose. California

Contents
1 Introduction, 484
2 Properties of X-Rays, 484
3 Classification of Radiopaques, 486
4 Heavy Metals and Their Salts, 486
4.1 Heavy Metals, 486
4.2 Heavy Metal Salts, 487
4.2.1 Barium Sulfate, 487
4.2.2 Bismuth Subnitrate, 489
4.2.3 Ferrites, 489
4.2.4 Metal Chelates, 489 .
4.2.5 Ultrasmall Ferrites, Gd-Chelates, and
MRI, 490
4.2.6 Tantalum Oxide, 492
4.2.7 Thorium Dioxide, 493
4.2.8 Other Metallic Salts, 493
5 Iodized Oils, 493
6 Organic Iodine Compounds, 495
6.1 Classification, 496
6.2 Synthesis, 512
6.2.1 Isomerism, 515
6.2.2 Potential Radiopaques, 516
6.3 Structure-Activity Relationship, 517
6.3.1 Substituent Structures, 517
6.3.2 Radiopacity, 531
6.3.3 Acidity, 535
6.3.4 Electron Density, 535
6.3.5 Hydrophilicity and Solubility, 536
6.3.6 Chemotoxicity, 536
6.3.7 Protein Binding and Excretion, 538
Burger's Medicinal Chemistry and Drug Discovery 6.3.8 Osmolality and Viscosity, 538
Sixth Edition, Volume 4: Autocoids, Diagnostics, 6.4 Analysis, 539
and Drugs from New Biology 6.5 Pharmacology, 540
Edited by Donald J. Abraham 6.5.1 The Cations, 541
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc. 6.5.2 Hyperosmolality, 542
483
484
6.5.3 Adverse Reactions and Toxicity, 543
6.5.4 Toxic Effects on Red Blood Cells, 547
6.5.5 Cardiovascular Effects, 550
6.5.6 Nephrotoxicity, 551
6.5.7 Neurotoxicity, 553
6.5.8 Protein Binding, 556
6.5.9 Histamine Release, 558
6.5.10 Pharmacokinetics, 559
6.5.11 Excretion, 562
6.5.12 Biotransformation, 565
6.6 Uses, 566
6.6.1 Computer-Assisted Tomography KT),
567
6.6.2 Spiral (or Helical) CT, 568
1 INTRODUCTION
Radiocontrast agents delineate body organs and
tissues against their immediate environment
during fluoroscopic or roentgenographic exami-
nation. They function by renderingthe spaces or
cavities occupied or the surfaces to which they
adhere either lucent or opaque in contrast to
their immediate surrounding tissues, in the
path of X-rays, and are accordingly classified as
negative- and positive-contrast agents (1, 2).
Positive-contrast agents absorb X-rays and pro-
duce a darker shadow on the fluoroscopic screen
and lighter or whiter shadows on the X-ray flm,
of the organ to be visualized against the sur-
rounding tissues (2). Radiopaques fall in this
category. Negative-contrast media are more
transparent than either water or body tissue to
X-rays, and give a lighter shadow on the fluoro-
scopic screen and a darker or blacker shadow on
the X-ray film. Fats, lipoid substances, and gases
such as air, oxygen, nitrogen, carbon dioxide, he-
lium, nitrous oxide, and xenon, absorbing less
X-ray radiation than the body tissue, belong to
this category.
Radiopaques have in common the property of
opacity to X-ray radiation and constitute many
substances that, although dissimilar in chemical
form, structure, and pharmacological proper-
ties, consist of elements of high atomic number.
To understand the unique dependency of ra-
diopacity on atomic number, a brief discussion
of the properties of X-rays is necessary.
2 PROPERTIES OF X-RAYS
In 1895 W. C. Roentgen discovered X-rays,
also known as Roentgen rays (3,4).Within a
Radiopaques
6.6.3 Ionics versus Nonionics, 568
6.6.4 Angiography, 569
6.6.5 Cholangiography, 569
6.6.6 Cholecystography, 570
6.6.7 Myelography, 570
6.6.8 Urography, 571
7 Miscellaneous Agents, 571
7.1 Perfluoroctyl Bromide, 571
7.2 Iodinated Particulate Suspensions, 572
7.2.1 Liposomes, 572
7.2.2 Emulsions, 575
7.2.3 Particulates, 576
7.3 Iodinated Polymers, 577
month of the discovery, medical examinations
were being made using the newly discovered
mysterious rays. X-rays are penetrating elec-
tromagnetic radiations with quantum ener-
gies of a few thousand to several million elec-
tron volts (eV), generated by transitions of
either bound electrons within an atom or free
electrons between two positive energy levels
in the field of an atomic nucleus (5). The
former is the atomic transition and consists of
the filling of a hole or vacancy in an inner shell
by an electron from an outer shell. The differ-
ence in binding energies between the two
shells involved is emitted as electromagnetic
radiation in the form of monochromatic (Le.,
monoenergetic) X-rays, typical of the element
and the transition. Transitions of free elec-
trons between energy states give rise to con-
tinuous X-rays; these are produced when elec-
trons accelerated to high kinetic energy are
allowed to impinge on a metal target. The
slowing down of fast electrons in the vicinity of
the target nucleus is essentially an electron
transition between two positive energy states
in the field of a target nucleus and leads to the
formation of bremsstrahlung ("braking radia-
tion"), composed of polychromic X-rays.
X-rays for medical use, produced by im-
pinging fast electrons on a tungsten target in
an X-ray tube, are polychromic. The diagnos-
tic X-rays have an energy range of 30 to 100
kVp (kilovoltsat the peak) but higher energies
(120-140 kVp) may be used in computed to-
mography (see Section 6.6.1). The energies of
X-rays produced in this manner depend on the
kinetic energy of the accelerated electrons (5).
2 Properties of X-Rays

Table 10.1 Physical Characteristics of Some Radiopaque Elements

Total Cross Section for X-rays of Several
Atomic K-Edge Energies," (cm2/g)
Number Density at
Element Z (g/cm3) (keV) 40 keV 60 keV 80 keV 100 keV
F 9 1.11 - 0.278 0.1912 0.1630 0.1497
Ca 20 1.55 4.038 1.792 0.6487 0.3630 0.2564
Ti 22 4.45 4.965 2.174 0.7543 0.4009 0.2706
Fe 26 7.86 7.112 3.559 1.174 0.5806 0.3643
Br 35 3.12 13.474 8.061 2.607 1.200 0.6849
Y 39 4.45 17.080 10.74 3.479 1.587 0.890
I 53 4.93 33.169 22.40 7.696 3.549 1.955
Ba 56 3.50 37.441 24.53 8.627 3.997 2.197
Gd 64 4.45 50.240 6.876 12.19 5.727 3.164
Yb 70 6.98 61.332 9.112 3.161 7.010 3.987
Ta 73 16.60 67.414 10.25 3.581 7.482 4.231
W 74 19.30 69.524 10.73 3.686 7.788 4.432
Pt 78 21.37 78.395 12.35 4.254 8.778 4.894
Au 79 19.37 80.723 12.81 4.442 2.111 5.116
Pb 82 11.32 88.006 14.05 4.893 2.335 5.461
Bi 83 9.80 90.527 14.98 5.267 2.520 5.808
Th 90 11.70 109.64 18.36 6.450 3.106 1.786
"Taken from Ref. 8.

In traversing through matter, X-rays are
attenuated by coherent (Rayleigh) and inco-
herent (Compton)scattering and are absorbed
by the photoelectric process (6, 7). X-rays of
energy below 100 keV are mainly absorbed by
the photoelectric process with a cross section
(i.e.,the probability for absorption) proportional
to Z5/E7I2(6),where E is the X-ray energy and Z
is the atomic number of the absorber. Energy
and wavelength of X-rays are related by the for-
mula A = 12.40/E7where h is in angstroms and E
in kiloelectron volts (8).As X-ray energy is in-
creased, absorption by the photoelectric process
diminishes, and Compton scattering becomes
important. At energies above 1.02 MeV, the X-
rays are predominantly attenuated by pair pro-
duction, creating electron-positron pairs; this
process does not play a role in the attenuation of
medical diagnostic X-rays.
The attenuation of a collimated beam of
monoenergetic X-rays is an exponential func-
tion of the depth of penetration (7). The rela-
tion may be expressed as
where Zo is the intensity of the initial X-ray
beam, I is the intensity of the transmitted X-
ray beam, e is the base of natural logarithm,
is the mass absorption (or attenuation) coeffi-
cient, and d is the mass of the material pene-
trated. The attenuation may also be expressed
using linear attenuation coefficient (pip),
where p represents the density, and x is the
thickness or the "mass-thickness" of the ma-
terial penetrated, equal to pd, as shown in .
Equation 10.la in the following:
The mass absorption coefficient is a func-
tion of the X-ray energy and the atomic num-
ber Z of the absorber and is equal to the total
cross section, expressed in cm2/g, of the inter-
actions mentioned above. In Table 10.1 are
listed the atomic number, density, K-edge, and
total cross section of elements that either have
been used or have potential use in contrast
media. The total cross sections given are cal-
culated for the absorption of monochromatic
X-rays of 4O,6O, 80, and 100 keV in energy (8);
for the absorption of polychromic X-rays of the
same energy designation, the values M e r some-
what. The total cross section decreases mono-
tonically as the X-ray energy is increased. At the
K and L absorption edges, sharp discontinuities
appear, and the total cross sections jump to
higher values as a result of enhanced absorption.

In roentgenography, the energy of X-rays is
optimized according to the depth of penetra-
tion desired, as determined by the size and
density of the object under examination (1,
9-11). Depending on the operational energy
range, a large fraction of the polychromatic
X-rays may fall in the region where absorption
by the radiopaque element is small or may fall
in the region of enhanced absorption near the
K-edge. The fact that iodine has a higher total
cross section for 40 keV X-rays than that of
tantalum or tungsten, for example, is attrib-
uted to the proximity of the X-ray energy to
the K-edge, which occurs at 33.169 keV. In-
creasing the iodine content of an iodinated
contrast medium from 28.7 to 37.5% doubles
the contrast of the radiographic image at an
X-ray energy of 70 kVp but increases the con-
trast by only 60% at 90 kVp (9).
Johns and Yaffe (12) used the energy and
mass dependency of the linear attenuation co-
efficient upon X-ray energy to characterize
normal and neoplastic breast tissues. Carroll
et al. (13, 14) showed that there are differ-
ences in the linear attenuation coefficients be-
tween normal and selected cancerous breast
tissues and that the contrast increases as the
beam energy in the energy range of 14 to 18
keV of monochromatic X-rays decreases.
These monochromatic X-rays were generated
at the National Synchrotron Light Source at
the Brookhaven National Laboratory.
Substances in medical radiography are
classified into five categories (2) according to
their opacity to X-rays:
Radiolucent: gases.
Moderately radiolucent: fatty tissues.
Intermediate: connective tissues, muscle,
blood, cartilage, cholesterol stones, uric
acid stones.
Moderately radiopaque: bone, calcium salts.
Very radiopaque: heavy metals and their
salts.
CLASSIFICATION OF RADIOPAQUES
The basic characteristics desirable for a con-
trast medium are (1)satisfactory radiopacity
(related to atomic number, material density,
and concentration), (2)stability, (3) pharma-
cological inertness, and (4) minimum sensitiz-
Radiopaques
ing properties (15). Radiopaques constitute
heavy metals and their salts, iodized oils, or-
ganic iodine compounds, and miscellaneous
agents, such as iodinated particulate suspen-
sions. Among these, the iodinated organic
compounds offer a wide range of diverse ra-
diopaque agents for clinical diagnosis in roent-
genography, and the heavy metal gadolinium
chelates can enhance the image contrast in
computed tomography and in nuclear mag-
netic resonance imaging.
4 HEAVY METALS A N D THEIR SALTS
4.1 Heavy Metals
Heavy metals with their high atomic numbers
have satisfactory radiopacity and are poten-
tially useful radiopaques. Powdered metals
such as tantalum, gold, and lead have been
used for experimental bronchography in dogs
(16-18). Tantalum elicits no unfavorable tis-
sue reaction and is widely used in surgery (19-
22). The cost of its application as a radiopaque
is minimal. Inhaled tantalum dust induces no
acute or chronic inflammatory response in the
airways or pulmonary tissue (23). Ingested
tantalum powder produces no untoward ef-
fects on the gastrointestinal (GI) tract and is
excreted. Injected tantalum powder is en-
gulfed by the Kupffer cells without visible
damage or pericellular reaction (24). The tox-
icity of tantalum powder is extremely low,
given that as much as 8000 mg/kg can be ad-
ministered orally without systemic toxicity
(17,22).
Nadel et al. (17, 25) were the first to use
tantalum dust for bronchography in canine
and human lungs. Tantalum dust, with an av-
erage particle size of 2.5 pm diameter, has
been administered by insufflations in bron-
chography, esophagography (251, and gastrog-
raphy (26). Tantalum dust adheres tena-
ciously to bronchial and esophagal mucosa, to
yield bronchograms and esophagograms of ex-
cellent detail over a prolonged period (17, 23,
25, 27). Experimental double-contrast cystog-
raphy has also been carried out (28). An aero-
sol preparation containing 2.4% tantalum
dust (particle size, 2-50 pm), 5% lecithin (as
surfactant), and 55% dichlorodifluorometh-
ane has been introduced to simplify the prep-
aration for the roentgenographic procedure
 4 Heavy Metals and Their Salts
and to shorten the preparation for exarnina-
tion (29). Commercial tantalum powders are
nonporous (30) and must be fractionated be-
fore use in bronchography (31). Because of its
high density and high atomic number, tanta-
lum compares favorably with other contrast
ii agents and yields superior bronchograms to
F those obtained with propylidone (15d)(17).
I Tantalum provides equal attenuation to an X-
ray beam with 115 to 1/10 as much as the
amount required of barium or iodine and
about 1/20 the amount required of iodized oils
(17,18). The metal is removed from the bron-
chi within a few days by the ciliary activity and
by coughing (17,23,32) and from the lung in a
much shorter time (23). The clearance of tan-
talum is slower, however, than that of barium
sulfate under similar conditions (23);this may
be attributed to its high density, which slows
the transport of particles by the flow of body
fluid. The clearance half-time ranges from 105
to 817 days when determined with the use of
radioactive ls2Ta (32).
Powdered tantalum (average particle size:
3 pm in diameter) has been given intrave-
nously as suspensions to dogs and rabbits for
splenohepatography (24).Although good sple-
nohepatograms have been obtained, the ag-
gregation of tantalum powder has caused fatal
pulmonary embolism in dogs. The usefulness
of tantalum dust for bronchography has been
reviewed (33,341.
Link et al. (35) proposed the use of tanta-
lum and tungsten to add radiopacity to hydro-
gel embolic agents for embolotherapy and
studied the emboli formed by coagulation un-
der high pressure of a 1:l or 2:l mixture of
tungsten particles (1-10 pm) and a liquid hy-
drogel (polyacrylonitrile copolymer) in a rab-
bit model.
4.2 Heavy Metal Salts
Soluble heavy metal salts are in general highly
toxic because of the presence of free metal
ions, although many insoluble ones including
oxides have been used in roentgenography
(36-42). Barium sulfate has been successfully
used as a contrast agent for visualization of
the alimentary tract for the past 70 years and
has not been supplanted by iodinated contrast
media (43). The ferrites are experimental ra-
diopaque agents because the magnetism of
this class of iron oxide allows the movement of
the ingested material to be controlled by an
external magnetic field (44, 45). Ultrasmall
superparamagnetic ferrite particles have clin-
ical use for contrast enhancement in magnetic
resonance imaging (MRI).
4.2.1 Barium Sulfate. Bachem and Giinther
(46) introduced barium sulfate in 1910 to re-
place insoluble and toxic bismuth salts for
clinical visualization of the alimentary tract.
Its nontoxicity, effectiveness, and low cost
have made barium sulfate the most widely
used radiocontrast agent for gastrointestinal
roentgenographic examination since that time
(47,48). Dihlmann and Hering (49) described
its usage up to 1993. Patton (50) reviewed the
history of barium sulfate as a contrast me-
dium and noted its commercial use in the early
days as a flour adulterant because of its non-
toxicity and lower cost.
Colloidal barium sulfate preparations are
available from numerous commercial sources.
Information about their exact composition has
not been freely available because of propri-
etary interest (47, 51). Preparations may dif-
fer in their effectiveness for coating mucosal
surfaces as determined by (1)particle size, (2)
ionic charge on suspended particles, (3) pH,
(4) resistance to flocculation, (5) suspension
aid, and (6) viscosity or osmotic toxicity (52).
The particle size of barium sulfate may
vary from a fraction of a micron to several
microns or more (48). Ultrafine grain size by
itself may give inferior visualization of the
gastric mucosa but the particles can be more
easily held in suspension. On the other hand,
particles larger than 1 pm may offer better
contrast, provided that they can be made to
stay in suspension. An electron micrograph of
barium sulfate particles of less than 1 pm in
diameter shows that they are of irregular
shape (53). The influence of microcrystalline
shape on the coating property of the suspen-
sion is not well studied.
The viscosity of barium sulfate suspension
is determined by the quantity and size of par-
ticles and can be modified by added peptizing
agents (52). A thick paste of barium sulfate in
water can be peptized or thinned by the addi-
tion of sodium citrate and sorbitol. The func-
tion of the citrate is to stabilize the colloidal
488
preparation and that of the sorbitol is to en-
hance that function. The use of a polybasic
acid and sugar alcohol combination can lead to
the incorporation of so much barium sulfate in
a liquid suspension as to obtain a preparation
with a specific gravity close to 3 (54,55).Other
polybasic acids such as tartaric acid and ethyl-
enediaminetetraacetic acid (edetic acid) may
also be used.
Particles in unprotected barium sulfate
suspensions have a tendency to aggregate, re-
sulting in flocculation. Such suspensions may
be stabilized by the use of a dispersingagent or
by placing an electric charge on the particle
(52). The surface of barium sulfate particles is
either positively or negatively charged, de-
pending on the residual lattice ions present. It
is also affected by the nature of the material
added to coat the particles. The coated parti-
cles are uniformly charged and flow easily on
account of the like charge on their surface,
which resists aggregation and increases the
colloidal stability.
Additives for coating barium sulfate parti-
cles include methylcellulose (47, 52, 53, 56-
61), carboxymethylcellulose (53, 54), hy-
droxypropylcellulose (53, 54, 62), chondroitin
sulfate (631, heparin (521, and sodium dextran
sulfate (52). Dispersing agents used for stabi-
lization and peptization of the colloidal sus-
pension include agar (48, 54, 551, acacia (47,
56), alginates (54, 56, 59, 64, 651, alginic acid
and propylene glycol mixtures (66), anionic
galactanes (66), bentonite (47,60),casein (471,
erythrobic acid (58, 59, 67, 681, gelatin (47,
60), glycerol (60, 69), gum arabic (53, 55, 621,
hexametaphosphate (57, 58, 621, lecithin (47,
65), mannitol (69), pectins (47, 54, 55, 62),
polyalkylarene sulfonates (66), polyethylene
glycol 400 (64, 69, 701, polymetaphosphate
(71), nonionic poly(oxyethylene)glycol stea-
rate (651, polysorbate 80 (47, 691, poly(viny1
alcohol) (54, 55, 70), poly(vinylpyrro1idone)
(54, 55, 62, 71), pyrophosphate (55), sodium
ascorbate (58,59,67-69), sodium dioctylsulfo-
succinate (69),sodium lauryl sulfoacetate (64,
69), sorbic acid (64, 691, sorbitol (54, 55, 64,
69), and trisodium citrate hydrate (55,64,66,
69). Classification of these agents by their
function is only superficial, given that many
have dual functions.
Radiopaques
The mobility of the particles depends on
the pH of the suspension, the ionic strength,
the age, and the method of preparation. Ad-
herence of barium sulfate particles to mucosal
membrane is pH dependent because the pH
affects not only the electrokinetic charge on
the barium sulfate particles but also the
charge on the mucosal lining, which is com-
posed of glycoprotein mucopolysaccharideand
is capable of carrying charges (52). Coating
of mucosal surfaces by commercial barium
sulfate preparations has been studied by
Schwartz et al. (72), who found that an opti-
mal viscosity is important for satisfactory
coating; at low viscosities the coating is too
thin, and at high viscosities the preparation is
too viscid for use. According to Fisher (43),
0.01 g of barium sulfate/cm2 of surface area is
needed to visualize mucosa.
Many different preparations of colloidal
barium sulfate for roentgenography are avail-
able commercially. The so-called barium
meals are not limited to liquid suspensions;
they also appear as tablets (73). Barium sul-
fate suspensions containing an effervescent
agent have been introduced for use in double-
contrast studies (74, 75). Barium sulfate may
also be coated with Fe,03, MgO, and A1,03
(63,761. The coated material has good dispers-
ibility and very low viscosity in acidic media.
Other barium preparations are also useful. A
barium titanate suspension was compared
with barium sulfate (72). An emulsified mix-
ture containing castor oil and barium sulfate
can visualize colon fistula more economically
than can iodinated contrast media (77).
Barium sulfate is used not only in roent-
genographic examination of the GI tract (51)
but also in inhalation bronchography (61).
Bullowa and Gottlieb (78) did early experi-
ments on dogs in 1920. Barium sulfate was
used for bronchography in infants and chil-
dren (79). According to Clement (61), satisfac-
tory bronchograms can be obtained with less
barium sulfate if the bronchial surface is pre-
viously exposed to methylcellulose. Methylcel-
lulose improves the bronchogram by forming a
viscous film on the bronchial mucosa to which
barium sulfate easily adheres, thus reducing
considerably the amount of contrast agent re-
quired for an examination.
4 Heavy Metals and Their Salts
When used in bronchography, barium sul-
fate is nonallergenic and nontoxic. It induces
only a mild, benign, foreign body reaction and
causes no pulmonary fibrosis or bronchial
spasms. It is cleared from the normal lung by
ciliary action, coughing, and phagocytosis at a
rate similar to that of tantalum (61).When the
bronchial surface is precoated with methylcel-
lulose, the clearance of barium sulfate is usu-
ally complete within 24-48 h (61). Any resid-
ual amount that may remain trapped in the
alveoli is usually located within the macro-
phages in tiny intra-alveolar granuloma.
When used in roentgenography of the GI tract,
barium sulfate has the inherent danger of in-
spissation and impaction when water is reab-
sorbed, particularly in the colon (48, 80, 81).
4.2.2 Bismuth Subnitrate. Bismuth subni-
trate was the first contrast agent used clini-
cally to visualize the alimentary tract (36). It
was found to be toxic in humans through the
reduction from nitrate to nitrite; as a result,
bismuth subcarbonate was substituted (82).
When the toxic action was traced to the metal
itself, the use of bismuth salts was discontin-
ued.
4.2.3 Ferrites. Novel materials such as fer-
rites were introduced as experimental radio-
contrast agents (44,45). Ferrites are iron ox-
ide (Fe,O,) in solid solution with one or more
metallic oxides. They possess about 80% of the
radiopacity of barium sulfate and a higher per-
centage if oxides of high atomic number are
incorporated. The ferrites are magnetic, a
property that allows their movement within
the body to be controlled by an external mag-
net. Because of their magnetic property, ul-
trasmall superparamagnetic iron oxide parti-
cles are studied in animals for accumulation in
lymph nodes, to develop a target-specific con-
trast agent for magnetic resonance imaging
(MRI) (83).
Frei et al. (44) reported the use of magne-
sium ferrite as a contrast material for X-ray
diagnosis. Sugimoto et al. (45) studied the
suitability of four ferrites containing Mg, Zn,
Cu, Ni, and Mn as radiopaques with respect to
toxicity, solubility in gastric juice, X-ray ab-
sorption, and effective magnetic field strength
necessary for controlling the ferrite powder.
These ferrites can be prepared to yield low sol-
ubility in acid or stimulated gastric juice. They
are nonallergenic and have no acute toxicity
(84). On prolonged daily oral administration
(30 days), the ferrites cause a slight decrease
in body weight, hematocrit, and the enzyme
glutamic-pyruvic transaminase in rats and
mice (85). The median lethal dose (LD,,) for
rats and mice is >20 and >10 gkg, respec-
tively (85).When fed to experimental animals,
the ferrites are cleared entirely from the body
within 1 week, as shown in absorption studies
using ,,Fe-, 54Mn-, or 65Zn-labeled ferrites
(84). Only soft ferrites, which do not coalesce
in the absence of the applied magnetic field,
are used as contrast media. Satisfactory roent-
genograms of the esophagus, stomach, bron-
chus, and small intestine have been obtained
with these ferrites (45). The particle size of the
ferrite powder may range from less than 1 mi-
cron to several microns in diameter, and a sta-
ble suspension of the particles may be ob-
tained using stabilizing and peptizing agents
(86-91) similar to those mentioned in Section
4.2.1.
4.2.4 Metal Chelates. Chelating agents ca-
pable of combining with metals of high atomic
number are potentially useful contrast media
'
(15, 37, 38). Chelation may significantly re-
duce the toxicity of metal ions; the toxicity of
Ca, Ni, Co, and Pb salts of ethylenediaminetet-
raacetic acid (EDTA or edetic acid) is many
times lower than the toxicity of the metal ions.
On the other hand, Cr, Cu, and Hg edetates
and their metal ions have comparable toxicity,
as indicated by the LD,, (7-day mortality) val-
ues. Because the binding between metal ions
and the ligands is reversible, the sequestered
metal ions in the latter group of chelates may
have become free in vivo.
In addition to edetic acid, multidentate
chelating agents such as 1,2-diaminocyclo-
hexane-N,Nr-tetraacetic acid, diethylenetri-
aminepentaacetic acid (DTPA), N,N1(2-hy-
droxycyclohexyl)ethylene-diaminediacetic
acid, 2-hydroxycyclohexylethylenediaminetri-
acetic acid, and P-hydroxyethylethylene dia-
minetriacetic acid have been used to sequester
heavy metal ions for roentgenographic pur-
poses (37). Solutions (8%)of Bi-DTPA and Pb-
EDTA were used in experimental bronchogra-

phy and angiography in dogs (37) but the
margin of safety between auseful dose and the
minimum lethal dose is so narrow as to render
them hazardous for clinical trials. Zwicker et
al. (92) compared iodinated and noniodinated
contrast media, that is, iopromide (2.94 molar
iodine, 370 mg I/mL) with gadolinium (as 0.5
molar Gd-DPTA, 78 mg GdJmL) and ytter-
bium (0.5 molar Yb-DPTA, 86 mg YbImL) in
computed tomography of aorta and liver in
dogs. At equimolar concentrations, Gd-DTPA
and Yb-DTPA are superior to iodine in maxi-
mum contrast enhancement of the aorta and
the liver. but iodine at 2.94 molar concentra-
tion exceeds these enhancement values of
metal chelates at 0.5 molar concentrations.
The lanthanide chelates, because of their high
atomic number and approximately 40%
greater efficiency in attenuatingx-rays of 120
kV than iodine at equivalent mass concentra-
tion, are useful agents for contrast enhance-
ment in computed tomography (CT) (93,94).
Schmitz et al. (94)found in a rabbit model that
the neutral macrocyclic chelate gadobutrol is a
more effective contrast agent than iopromide
-
for CT at lower doses of the imaging atom.
These agents may be indicated in patients
with known previous allergic reactions to io-
dinated contrast media.
The high atomic number and the large
magnetic moment of gadolinium have made
its compounds potentially useful for both CT
and nuclear magnetic resonance imaging
(MRI). The effect of gadolinium chelates on
the relaxation behavior of water protons pro-
vides the contrast in the MRI of human tissues
(95). The mechanisms for contrast enhance-
ment by Gd-chelates in CT and in MRI are
very different, although the imaging results
are very similar. A brief description of MRI is
given below as a basis for discussion of the
development and use of potential ferrites and
gadolinium contrast-enhancing agents.
4.2.5 Ultrasmall Ferrites, Gd-Chelates, and
MRI. Nuclear MRI is dependent on the use of
superparamagnetic and paramagnetic materi-
als to enhance the contrast in tissue discrimi-
nation and is both complex and difficult. The
patient is not exposed to X-ray radiation while
undergoing MRI. Unlike the radiocontrast
media attenuating the X-rays, these agents fa-
Radiopaques
cilitate the relaxation of water-protons and
are not visualized directly in the MRI image
(96,97). In MRI, the patient is placed within a
uniform magnetic field; the water-protons in
the body of the patient will line up, giving a net
nuclear macroscopic magnetization along the
long axis of the patient. If one applies an ex-
ternal radiofrequency pulse to induce mag-
netic resonance, the nuclear magnetization of
the individual protons will relax them back to
their original position in an exponential man-
ner, called spin-lattice relaxation, with a time
constant TI. Relaxation also occurs in the
transverse axis, called spin-spin relaxation,
with a time constant T2. The MR contrast
agents that facilitate the relaxation will in-
crease or decrease the signal intensity accord-
ingly. For diagnostic purposes, relaxation data
acquisition after a pulse sequence is computer
controlled and the data are reconstructed and
displayed as an image, to show the contrast
enhancement between normal and diseased
tissues. According to Carr (98),the MR images
are dependent on pulse sequences, proton den-
sity, TI, and T2. Saturation-recovery se-
quences rely on proton density, inversion-re-
covery sequences on proton density and TI,
and spin-echo sequences on proton density
and T2.
The contrast media for MRI, according-to
their effect on image enhancement, are cate-
gorized as (1)relaxivity-based T1 agents and
(2) susceptibility-based T2 agents (96, 97).
The term l/Ti,where i = 1,2, represents the
rate of relaxation of solvent nuclei. Relaxivity
(RJ is obtained as the slope of relaxation rate
per 1 mM of paramagnetic species, in the unit
of mM-' s-'. It represents the efficiency with
which the contrast agent enhances the proton
relaxation rate of water and is defined as fol-
lows (94):
where the suffixes p and 0 indicate with and
without paramagnetic species, respectively.
Susceptibility effect refers to the relaxation of
T2, attributed to the moving spins relaxed by
the fluctuating field. A paramagnetic metal
complex can interact strongly or weakly with
water molecules, depending on whether these
4 Heavy Metals and Their Salts
water molecules are covalently bonded or hy-
drogen bonded to the metal complex, some-
times referred to as "inner-sphere" relax-
ation, or whether the water molecules are
passing through the field by translational dif-
fusion, often referred to as "outer-sphere" re-
laxation (95, 96). A shortening of the T1 and
T2 relaxation times will lead, respectively, to
either a positive (brightening) or a negative
(darkening) effect in image intensity. Metal
ions are toxic and need to be sequestered as
chelates with very small dissociation constant
for safe use as contrast media. Chelation de-
creases the relaxivity.
Ultrasmall ferrite particles and lanthanide
chelates are potential contrast media in MR
imaging. Ferromagnetic, superparamagnetic,
and paramagnetic compounds affect 1/T1 and
1lT2 differently (99). Ferromagnetic and su-
perparamagnetic iron oxide particles enhance
1lT2 more than 11T1and have a greater trans-
verse relaxivityflongitudinal relaxivity ratio
-
(R2/R1 >> 1) than paramagnetic substances
(R2/R1 1) (99). The iron oxide particles,
when prepared by different methods, differ in
particle size, polycrystalline state, core con-
centration, and the dextran coating. The rec-
ognition of these iron oxide particles by mac-
rophages and their distribution in the body
are determined by their size and surface prop-
erties (100). The ultrasmall particles can mi-
grate across capillary endothelium upon intra-
venous administration and are taken up by
the reticuloendothelial system, including the
liver, spleen, lymph nodes, and bone marrow.
Ferrite particles AM1 25 are about 80 nm in
-
diameter, have a very short blood half-time of
6 min, and are phagocytosed by Kupffer cells
in the liver, resulting in contrast enhance-
ment between normal liver and tumor metas-
tasis (101, 102). The ultrasmall superpara-
magnetic iron oxide particles (USPIO),
obtained by size fractionation of AM1 25, have
a mean diameter of 11 nm and a plasma half-
time of 81 min and tend to accumulate in
lymph nodes (83,103). USPIOs have a molec-
ular weight of approximately 700 kDa by gel
filtration, comparable to that of ferritin (11
nm; molecular weight, 440-600 kDa) (103).
AM1 227 is a nanoparticulate iron oxide with a
mean size of 18 nm (17-21 nm) and an outer
coating of low molecular weight TI0 dextran
(104, 105). BMS 180549 (Squibb Diagnostics,
Princeton, NJ) is similar to AM1 227 in size
but has an iron core of 4.3-6.0 nm in diameter
and a blood half-life of greater than 200 min,
and is clinically useful for differentiating reac-
tive from tumor-bearing lymph nodes (106,
107). The maximum dose is 1.7 mg Fekg or
120 mg Fe for a 70-kg person, which is small
compared to the body iron store. Intravenous
or interstitial administration of superpara-
magnetic particles is relatively free of unto-
ward side effects, but clumping of the particles
and microembolization of the AM1 25 particles
may lead to back pain and fever (108). The
compound is nontoxic and readily metabolized
in the body. Its cellular uptake and trafficking
is by receptor-mediated endocytosis to the ter-
minal lysosome
- (109). Ultrasmall iron oxide
particles can be targeted to asialoglycoprotein
receptor in MR receptor imaging (103). An
oral formulation of magnetic ferrite particles
was also tested for enhancing visualization of
the GI tract in MR imaging (110).
Gadolinium has seven unpaired 4f elec-
trons and a large magnetic moment and is
highly paramagnetic. The free gadolinium
ions are toxic. When administered intrave-
nously, the ions combine with endogenous
metal-binding sites and endogenously avail-
able counter ions, such as phosphate, car-
'
bonate, and hydroxide, to form insoluble
complexes that are very slowly excreted. Se-
questering the metal ions by chelation with
organic chelating agents decreases the toxic-
ity. Gd-DTPA meglumine salt, a chelate of
gadolinium with diethylenetriaminepentaace-
tic acid (DTPA), was reported to have a me-
dian lethal dose (LD,,) of 10 mmolkg, com-
pared to that of gadolinium chloride (GdC1,)
with an LD,, value of 0.5 mmol/kg in the rat
when administered intravenously (110). Gd-
DTPA has a thermodynamic stability constant
of 1022.46at 25°C (94). Gd-chelates enhance
contrast in MRI. The signal response of gado-
linium is not dose dependent but biphasic;
that is, the signal intensity increases initially
and then decreases with an increase in Gd con-
centration (112). Paramagnetic gadolinium-
chelated complexes for MRI (113) can be cate-
gorized, based on their chemical structures,
into two classes: (1)those sequestered by lin-
ear ligands (EDTA, DTPA, etc.) and (2) those

sequestered by macrocyclic ligands (polyaza
polycarboxylic acids, etc.) as follows:
1. Gadolinium-chelates with linear ligands
0 Gadobenate dimeglumine (Gd-BOPTA)
0 Gadodiamide (Gd-DTPA-BMA)
0 Gadopenamide
0 Gadopentetate dimeglumine (Gd-DTPA)
0 Gadoversetamide
0 Gadoxetic acid
2. Gadolinium-chelates with macrocyclic li-
gands
0 Gadobutrol
0 Gadoteric acid (Gd-DOTA)
0 Gadoteridol (Gd-HP-D03A)
Tweedle et al. (114)studied the biodistribu-
tion of gadodiamide, gadopentetate, gadoter-
ate, and gadoteridol, labeled with radioactive
gadolinium-153 in both mice and rats. Ga-
dodiamide and gadopentetate have linear li-
gands, and gadoterate and gadoteridol have
macrocyclic ligands. Gadodiamide and gado-
teridol are nonionic, whereas gadopentetate
and gadoterate are ionic. These authors found
that the radioactivity of the linear complexes,
compared with the macrocyclic complexes, re-
mained longer in the bone and liver of the an-
imals. The order of their residual Gd concen-
tration at long residence times from the lowest
-
to the highest, was gadoteridol gadoterate 5
gadopentetate << gadodiamide. The macrocy-
clic chelates of gadolinium have higher ther-
modynamic stability constants and tend to be
more stable and do not undergo decomplex-
ation in vivo. Gd-DTPA is rapidly excreted
through the kidneys. Dean et al. (115) listed
the rank of tissue distribution of Gd-DTPA in
the rat 2 min after intravenous injection of the
contrast agent. McLachlan et al. (116) showed
the mean distribution half-life of the injected
gadoteridol in human volunteers to be 0.20 2
0.04 h and the mean elimination half-life 1.57
2 0.08 h. More than 94% of the contrast agent
was excreted in the urine in 24 h. The elimi-
nation half-life and the distribution half-life of
gadoteridol were independent of the dose
used. Staks et al. (117) studied the phannaco-
kinetics of gadobutrol in humans at different
dose levels, up to the maximum dose level at
Radiopaques
0.5 mmolkgbody weight, and showed that the
contrast agent distributed predominantly in
the extracellular fluid space, with a half-life in
plasma of approximately 1.5 h. More than 95%
of the injected dose was excreted by glomeru-
lar filtration within 12 h. No metabolite was
detected.
Paramagnetic contrast agents that are dis-
tributed throughout the extracellular space
and rapidly excreted by the kidneys have lim-
ited value for perfusion and organ function
studies in MRI. Brasch et al. (118,119) showed
that human serum albumin covalently bound
to 5-18 Gd-DTPA molecules were useful for
blood-pool enhancement but these macromol-
ecules were slowly excreted. These authors
also quantified capillary permeability of ex-
perimental mammary adenocarcinoma in fe-
male rats with albumin-(Gd-DTPA),, (120),
and identified and differentiated pulmonary
diseases with polylysine-(Gd-DTPA),, (121).
Paramagnetic dextran (i.e., dextran linked to
Gd-DTPA by diaminohexane through carba-
mate and carboxymethyl groups) was pre-
pared as a potential MRI contrast agent (122).
Liposomes containing Gd-HP-D03A or am-
phipathetic agents were evaluated for use in
liver MR imaging (123, 124). Gadozelite, a
gadolinium zeolite complex, has been listed for
oral use (113). New derivatives of linear and
macrocyclic polyaza and polyamino polycar-
boxylic acids have been synthesized as ligands
for potential MRI contrast agents. Among
them are Gd-DTPA-bis(arnide)complexes, 15-
to 17-membered macrocyclics with three pen-
dent carboxymethyl groups, and others (125-
131). Ranganathan et al. (132) designed and
synthesized for testing biochemical processes
a series of multimeric MRI agents, with molec-
ular weight of 1-5 kDa and with a varying
number of hydrated water molecules in the
inner sphere of the gadolinium atom to modu-
late the reflexivity.
4.2.6 Tantalum Oxide. Although tantalum
oxide is less radiopaque than tantalum powder
by a factor of 0.5 per volume or 0.8 per mass,
its chemical and biologic inertness and toxicity
are very similar to those of tantalum (40). The
technical advantages and safety in handling
and storage of the oxide make it superior for
use in roentgenography to metallic tantalum,
5 Iodized Oils
which presents an explosion hazard. The oxide
is formed as P-T%O,, with a particle size of
about 1 mm or less, by burning metallic tanta-
lum powder in air. It is used in bronchography
and esophagography by topical application.
When injected intravenously in dogs and rab-
bits in experimental hepatosplenography, the
oxide forms microscopic emboli in the liver
and kidney. Injected particles of the oxide are
engulfed by the reticuloendothelial cells with-
out appreciable cellular or pericellular reac-
tion.
4.2.7 Thorium Dioxide. The use of tho-
rium dioxide as a contrast agent began in 1928
to 1929 (133);it has been used under the name
of Thorotrast (Fellows-Testager Division, De-
troit, MI) for angiography and cerebral arte-
riography. Thorotrast is a colloidal suspension
of 25% by weight of thorium dioxide, of parti-
cle size less than 0.15 pm diameter, prepared
by oxidation of thorium oxalate at 550°C (134).
The high atomic number, high density, and
the lack of acute toxicity make thorium diox-
ide an ideal radiopaque for use in large quan-
tities in roentgenography (135). Thorium di-
oxide gives excellent contrast and has proved
to be valuable in studies in which other con-
trast media would fail.
The drawback of using thorium dioxide as a
contrast agent is its radioactivity and indefinite
retention in the body (136,137).Thorium-232 is
the longest-lived parent radionuclide in the tho-
rium series (138). A considerable amount of
work has been done regarding the disposition
and fate of thorium dioxide after its systemic
and local use in humans for roentgenography
(139-147). The dioxide when administered in-
travenously is engulfed by the phagocytic cells
of the reticuloendothelial system and is per-
manently localized in these cells (139). The
long-term effects of radiation are fibrosis and
neoplastic growth in the liver and spleen and
fibrosis of their efferent lymph nodes (39,
146). Localization of thorium and its daughter
nuclides in the bone may result in leukemia
and other blood dyscrasias (144). Locally in-
jected thorium dioxide, if in contact with epi-
thelium for long periods, induces carcinoma
(39, 148).
When colloidal thorium dioxide was applied
within the lumen of the GI tract, no deleteri-
493
ous late effects were observed (39). The con-
tinuous shedding of the intestinal epithelium
can remove the deposited dioxide and excrete
it together with the intestinal content.
Because of the radiation effects of thorium,
the use of thorium dioxide is limited only to
the procedures approved by the U.S. Food and
Drug Administration, such as hepatolienogra-
phy in patients with metastatic cancer (39).
4.2.8 Other Metallic Salts. In addition to
the heavy metal salts mentioned above, bar-
ium titanate and barium metatitanate have
been used for visualization of the hypophar-
ynx, esophagus, stomach, and small intestine
(41, 149). Barium metatitanate is used as a
powder with grains measuring from 0.8 to 1.0
pm. It is insoluble in water and has a density
of 6 compared to that of barium sulfate with a
density of 4.5. In addition, the metatitanate
adheres better than barium sulfate to the in-
testinal mucosa, a property considered favor-
able for visualization. Powdered calcium tung-
state can also be used to opacify airways but
was found ineffective for filling airways
smaller than 2 mm in diameter (42).
5 IODIZED OILS
Radiopaque iodine atoms can be introduced
into vegetable oils to form iodized oils by
.
reaction with hydroiodic acid. This converts
unsaturated fatty acid groups into iodinated
saturated ones, such as linoleic acid to diio-
dostearic acid and oleic acid to monoiodoste-
aric acid (150). Commercial preparations of io-
dized oils are glyceryl esters (Lipiodol) and
ethyl esters (Ethiodol) of iodinated fatty acids
of poppyseed oil. Lipiodol and Ethiodol con-
tain approximately 38-42 and 37% of organi-
cally bound iodine, respectively. Ethyl diio-
dostearate (45.0% I) and ethyl triiodostearate
(55.2%I) have higher iodine content than that
of Ethiodol and are available as emulsions
(151). The iodized oils are yellow or amber and
decompose with liberation of iodine upon ex-
posure to air or light. Ethiodol has a lower
viscosity than that of Lipiodol. The toxicity or
tolerance of iodized oils is determined by their
viscosity (150).
Iodinated oils are emulsified for injection
with surfactants such as polyoxyethylene sor-
494
bitan monooleate (Tween-80) and sorbitan
monooleate (Span-80) (152), polyethylene gly-
col-400 stearate (153,1541, soya lecithin (155),
or ethylene oxide and castor oil (156),and sta-
bilized with polyhydric alcohols such as glyc-
erol and glucose. The toxicity of iodized oils
administered intralymphatically is between
one and two times that administered intrave-
nously (150, 157). Ethiodol has an average le-
thal dose in the dog of 3.62 mL/kg for intra-
lymphatic injection compared to 1.58 mL/kg
for intravenous injection (157). Iodized oils
are injected slowly into the peripheral lym-
phatic; an 8-mL dose is usually given over a
period of 40-80 min at a pressure of 0.4 atm.
High injection speeds and pressure may cause
rupture of the lymph trunk and extravasation.
Intralymphatically injected oil contrast
medium may remain in the lymph nodes for
months or even for more than 1 year (150).
The excess of that retained in the lymphatic
system will enter the systemic veins through
the thoracic duct or by way of lymphaticov-
enous communications and eventually pass
into the pulmonary artery and its branches, to
be distributed in the lung capillaries. Experi-
ments in the dog showed that 50% of the in-
jected Ethiodol was found in the lung and
about 23%in the nodes at 3 days post-lymphog-
raphy. The concentration of iodized oil in the
nodes remained essentially the same but that
in the lung had decreased to about 13% of the
dose at 17 days postinjection (150, 158).
Metabolic studies with l3'I-labeled Ethio-
do1 indicated that the iodized oil was rapidly
deiodinated by enzymes in tissues with the io-
dine appearing as inorganic iodide, which was
excreted by the kidney. In humans, no more
than 0.5% of the injected iodized oil was found
in the blood at any one time, and the urinary
excretion was less than 2.5% of the dose per
day (159). The most serious side effect of the
iodized oils is pulmonary or systemic emboli-
zation and granuloma formation (1601, which
is related to the particle size of the oily drops
(150, 157), but Kupffer cells can actively cap-
ture and phagocytize the iodized oil droplets
(161).
Iodized oil is used in lymphography (150,
157),intra-arterial hepatography (1541, intra-
venous hepatosplenography (162), and hys-
terosalpingography (1601, and also used for
Radiopaques
treatment of hepatic tumors by chememboli-
zation (161). In the latter the iodized oil addi-
tionally exerts a synergistic effect by activat-
ing the immune system in the liver. The oily
contrast medium allows the detection of small
avascular masses in the liver or spleen by com-
puted tomography, which is superior to both
contrast angiography with water-soluble con-
trast media and scanning with radiopharma-
ceuticals; the former is too transient for tomo-
graphic imaging and the latter is inadequate
in resolution (162). Miyamoto et al. (160)
found that the water-soluble nonionic iodin-
ated contrast agent iotrolan has none of these
side effects.
Lipiodol, when injected intra-arterially,
can diagnose malignant hepatic tumors (163).
Lipiodol as a carrier for anticancer drugs, such
as neocarzinostatin, mitomycin, aclarubicin,
and doxorubicin, has been administered to pa-
tients with hepatomas (164). In hysterosalpin-
gography both oil and aqueous contrast media
can reliably provide information about uter-
ine-tubal anatomy, although Ethiodol gives a
sharper image and the added advantage of a
high conception rate (165-167). After oil con-
trast medium hysterosalpingography, one-
third of the infertile women had normal preg-
nancies and childbirths (167, 168). An in vitro
study showed that a potential mechanism for
fertility enhancement might be attributed30
the inhibition of peritoneal lymphocyte and
macrophage function by the oil contrast me-
dium. The advantages of Ethiodol versus dia-
trizoate, ioxaglate, and iohexol in hysterosal-
pingography have been varied (167).
Kishi et al. (169) evaluated the acute toxic-
ity of Lipiodol infusion into the hepatic ar-
teries (HAI) of beagles and found the influ-
ence of Lipiodol HA1 to be dose dependent.
The infused Lipiodol first passed through an
arterioportal communication and distrib-
uted through the hepatic sinusoids to pulmo-
nary capillaries and thence into systemic
blood circulation. The circulation and emboli-
zation of oil droplets were found in the renal
tubular cells of supracapsular cortex, the cho-
roid plexus, the vascular endothelium, and the
pancreatic duct epithelium, showing a process
of intracellular collection of Lipiodol from the
systemic blood circulation and of further me-
tabolism-provokingcellular reactions.
6 Organic Iodine Compounds
6 ORGANIC IODINE COMPOUNDS
Organic iodine compounds make up the larg-
est group of radiopaques or radiocontrast
agents used in roentgenographic examination.
The high atomic number of the iodine atom
confers radiopacity on these molecules. These
agents may be classified according to their ap-
plication as angiographic, cholegraphic, uro-
graphic, and myelographic agents, or accord-
ing to their route of administration as oral,
intravascular, or intrathecal agents. They are
used in high concentrations for better image
resolution. An estimate in 1993 put the use of
these agents as diagnostic aids on a worldwide
basis at a market value of over $2 billion per
annum (170). In the 1970s, the annual con-
sumption of this group of agents in the United
States surpassed 2000 metric tons and, even
with the advent of computed tomography, the
aggregate consumption of these media was
still about 1400 tons as late as 1990 (171).
Grainger (172) in 1982 traced the early his-
tory of development of intravenous contrast
media and the initial use of pyridine com-
pounds containing iodine as radiocontrast me-
dia. Binz (173) found this group of iodinated
pyridine compounds to be excreted by the kid-
ney and the liver in substantial concentration
and therefore labeled them the "Selectan
group," to indicate their selectivity. Some of
them were used for retrograde cystography
and pyelography. Graham et al. (174) in 1926
used tetraiodophenolphthalein as the first
contrast agent for intravenous cholecystogra-
phy. This compound was widely used for gall
bladder visualization until 1940, when Dohrn
and Diedrich (175) introduced iodoalphionic
acid and produced a better roentgenogram of
the gall bladder and fewer adverse reactions;
this remained the agent of choice until the
early 1950s. At this time a large variety of io-
dinated organic radiocontrast agents, based
on the 2,4,6-triiodobenzoic acid moiety, were
introduced, most of which gave even better
visualization and fewer side effects.
Wallingford (176) conceived the idea of us-
ing the benzene ring as carrier for iodine at-
oms. Strain (36), Grainger (172), A l m h (177,
178), and Sovak (171) reviewed the historical
development of organic iodine compounds as
radiopaque agents, including the past, the
present, and the future trends in the develop-
ment of iodinated contrast media. Archer
(179) discussed the chemical aspects of some
cholecystographic agents developed before
1959, including iopanoic acid. Hoey et al. (180)
compiled a list of iodinated compounds syn-
thesized up to 1971 as X-ray contrast media to
correlate their structure-activity relationship.
From clinical experience and theoretical con-
siderations, Alm6n (178, 181, 182) suggested
the use of nonionic contrast agents and postu-
lated that the nonionic contrast agents would
be less toxic than the ionic ones because of
lower osmolality. This theory stimulated the
search for nonionic iodinated compounds,
which included the synthesis and testing of
monomers (183-186), bis compounds (9,187-
1901, and polymers (191,192) as improved ra-
diopaques. The outcome of such studies
brought forth the first-generation nonionic
monomer metrizamide (183,184),the second-
generation nonionic monomers iohexol(193),
iopamidol (194-196), and so forth; the non-
ionic bis compounds iotrolan (1971, iodixanol
(198), and so forth; and the ionic, monoacidic
bis compound ioxaglate (199, 200). Hoey and
Smith (201) compiled a comprehensive list of
ionic and nonionic monomers and bis com-
pounds synthesized up to 1984, and reviewed
and discussed the chemistry and physico-
chemical properties of the contrast agents as
well as the safety and toxicity of different sub-
stituent groups. Newer drug design theories
have focused on searching for safer and more
effective contrast agents by masking the hy-
drophobic regions of the triiodophenyl nucleus
with small alkyl chain polyhydroxyalkyl
groups to increase hydrophilicity, to minimize
protein interaction, and to reduce toxicity
(171,182,202-205). The more powerful imag-
ing techniques, such as computer-assisted to-
mography (CT), delayed CT, dynamic CT, spi-
ral (or helical) CT, and digital subtraction
angiography (DSA),depend on safer and more
cost-effective contrast media to obtain ana-
tomic and organ function information. The
progress in clinical three-dimensional imaging
has been reviewed (206-208).
In subsequent sections, the iodinated ra-
diopaques are discussed with respect to their
classification, synthesis, structure-activity re-
lationships, analysis, pharmacology, and uses.
496
The section on pharmacology includes the top-
ics of cations, hyperosmolality, adverse reac-
tions and toxicity, toxic effects on red blood
cells, cardiovascular effects, nephrotoxicity,
neurotoxicity, protein binding, histamine re-
lease, pharmacokinetics, formulation, excre-
tion, and biotransformation.
The subject has been discussed earlier by
Hoppe (209, 210), and Ackerman (211) and
reviewed from the point of view of drug design
by Herms and Taenzer (212). A number of
monographs, from conference proceedings on
various aspects of iodinated contrast media
with specific information, have appeared (10,
213-218). Sovak (171) described briefly the
history of the development of contrast media
and his role in the search for an ideal agent.
6.1 Classification
In general, iodinated contrast agents contain
either the pyridone or the benzene nucleus as
the iodine carrier. With a few exceptions,
newer contrast agents are derivatives of 2,4,6-
triiodobenzoic acid and its congeners (1).The
R (solubility)
I
(opacity) I I (opacity)
detoxifying)
XY
(solubilizing,
I
Y (solubilizing,
(opacity)
detoxifying)
adoption of the benzene ring to replace the
pyridone nucleus as the iodine carrier for io-
dinated contrast agents originated from the
observation by Wallingford et al. (219, 220)
that iodobenzoic acids have very low toxicity
and that the substituent groups determine the
molecular and pharmacological properties.
Acylation, substitution, and triiodination can
further reduce the toxicity of the iodobenzoic
acid neucleus (201).
Iodinated contrast agents may be classified
as ionic and nonionic, monomers and bis com-
pounds. The term dimers is often used to refer
to the bis compounds. The classification is
based on whether the contrast molecule con-
tains one or two 2,4,6-triiodophenyl rings and
Radiopaques
an ionizing group. In an ionic contrast agent
the R group in (1)represents a strongly hydro-
philic functional group such as carboxyl, al-
kyl-, aralkyl- or alkoxyalkylcarboxyl group,
and so forth. These groups show both hydro-
philic and lipophilic properties. The X and Y
groups are hydrophilic and exert a strong in-
fluence on both pharmacological properties
(such as chemotoxicity, biological tolerance,
and biotransformation) and physicochemical
properties (such as solubility, osmolality, vis-
cosity, and protein binding). In a nonionic con-
trast agent, the R, X, and Y groups are short-
chain highly hydrophilic groups, in the form of
2-hydroxyethyl, 1,3-dihydroxypropyl, 2,3-di-
hydroxypropyl, or D-glucopyranosyl, and so
forth. These substituent groups are linked to
the phenyl ring by coupler groups, such as
amide, reversed amide, amino, or others (201).
Atoms of the coupler groups linked to the 1,3,
and 5 positions of the triiodinated phenyl ring
can be expressed in designated notations as
CCC, CCN, CNN, CNH, NNN, and so forth, to
indicate the nature of the starting material
and the ring electron density. This convention
was first introduced by Gries and Miitzel(221)
and will be adopted here to indicate subclassi-
fication of the iodinated contrast agents by
structure.
In the following, contrast agents that have
been accepted for clinical use are listed'by
their United States Accepted Names (USAN)
(222) or by their International Nonpropri-
etary Names (INN). No proprietary names of
the contrast agents will be given. For a listing
of the proprietary names, chemical formulas,
and uses, the International Drug Directory
(223) and early compilations by Knoefel (224)
and Strain and Rogoff (225) should be con-
sulted. Fischer (226) has catalogued, up to
1986, current higher and lower osmolality in-
travascular contrast media (abbreviated as
HOCM and LOCM, respectively) and listed
their iodine content (milligrams of iodine per
milliliter of fluid), osmolality, viscosity, and
available dosage form sizes, by use of informa-
tion obtained from manufacturers.
Classifications of ionic and nonionic mono-
mers and dimers or bis compounds are shown
with their structures in Table 10.2. Table 10.3
contains the chemical names of these ra-
diopaque agents and available properties;
6 Organic Iodine Compounds 497

Tab:,? 10.2 Classification and List of Names and Formulae of Contrast Agents
A. Ionic monomers
1. Triiodobenzoates (2) Iophenoxic acid (b)
Acetrizoate (a) Ioprocemic acid ( c )
Diatrizoate (b) Ipodic acid (d)
Diprotrizoate (c) Tyropanoic acid (e)
Iodamide (d) Iolidonic acid (f)
Iodamide meglumine Iomorinic acid (g)
Iotrizoic acid (e) 4. Triiodophenoxy alkanoates
Ioxotrizoic acid (f) Iolixanic acid (h)
Metrizoate (g) Iopronic acid (i)
2. Triiodoisophthalamates (3) Iobutoic acid Cj)
Ioglicic acid (a) 5. Triiodobenzamides (5)
Ioseric acid (b) Iobenzamic acid (a)
Iothalamic acid (c) 6. Triiodoanilides (6)
Ioxitalamic acid (d) Iocetamic acid (a)
3. Triiodophenyl alkanoates (4) Iomeglamic acid (b)
Iopanoic acid (a) Iosumetric acid (c)
B. Nonionic monomers
1. Triiodo-1,3-benzenedicarboxamides(7) Ioxilan Cj)
Iobitridol (a) 3H2-Iopiperidol-A(k)
Iohexol (b) 2. Triiodophenyl-D-gluconoamides (8)
Iomeprol (c) Ioglucol (a)
Iopamidol (d) Ioglucomide (b)
Iopentol (e) Ioglunide (c)
Iopromide (f) Iogulamide (d)
Iosimide (g) Iosarcol (e)
Iotriside (h) Metrizamide (f)
Ioversol (i)
C. Ionic dimers or bis compounds
1. Bis-triiodobenzoates (9) Iosulamide (f)
Iocarmic acid (a) Iotetric acid (g)
Iodipamide (b) Iotranic acid (h)
Iodoxamic acid (c) Iotroxic acid (i)
Iodoxamate meglumine Ioxaglic acid Cj)
Ioglycamic acid (d) Ioxaglate meglumine
Iosefamic acid (e) Iozomic acid (k)
D. Nonionic dimers or bis compounds
1. Bis-triiodo-1,3-benzenedicarboxamides(10,lO')
Iodecol (a)
Iodixanol (b)
Iotasul (c)
Iotrolan (or iotrol) (dl
Iofratol (e)
E. Miscellaneous
1. Other dimer and polymer Iopydol (c)
Ioxabrolic acid (1 1) Propyliodone (d)
Tris(iothalamic acid) (12) Sodium iodomethamate (Iodoxyl) (e)
2. Diiodophenyl alkanoate 5. Iodophthaleins
Iodoalphionic acid (13) Iodophthalein (16)
3. Iodophenyl alkanoate 6. Others
Iophendylate (14) Methiodal sodium (17)
4. Diiodopyridones (15) Dimethiodal sodium (18)
Iopydone (a) Iodohippurate sodium (19)
Iodopyracet (b)
 Radiopaques

Table 10.2 (Continued)

COOH
I

CNH -NHCOCH3 -H
CNN -NHCOCH3 -NHCOCH3
CNN -NHCOCH2CH3 -NHCOCH2CH3
CNC -NHCOCH3 -CH2NHCOCH3
CNH -NHCOCH2[OCH2CH2140CH3 -H
CNN -NHCOCH3 -NHCOCH20H
CNN -NHCOCH3 -N(CH3)COCH3
COOH
I

(3)
(3) 'MJe R1 R2
a CCN -CH2CONHCH3 -NHCOCH3
b CCN -CH(CH20H)CONHCH3 -NHCOCH20CP3
c CCN -CH3 -NHCOCH3
d CCN -CH2CH20H -NHCOCH3
XCOOH

CNH
COH
CNH
CNH
CNH
CNH

CNH
ONH
ONH
OCH
6 Organic Iodine Compounds

Table 10.2 (Continued)

COX
I

a CNH -N(C,H5)CH2CH2COOH -NH2 -H

(6)
(6) 'be X R1 R2
a NNH -CH2CH(CH3)- -COCH3 -NH2
b NNH -CO(CH2),- -CH3 -NH2
c NNH -CO(CH,),- -CH2CH3 -NHCH3

(7)
(7) Type R1 R2
R3 R4 R5
a CCN -CH2CHOHCH20H -CH3 -CH2CHOHCH20H -CH3 -NHCOCH(CH,OH),
b CCN -CH2CHOHCH20H -H -CH2CHOHCH20H -H -N(COCH3)CH2CHOHCH20H
c CCN -CH2CHOHCH20H -H -CH2CHOHCH20H -H -N(CH3)COCH20H
d CCN -CH(CH20H)2 -H -CH(CH20H)2 -H -NHCOCH(OH)CH3"
e CCN -CH2CHOHCH20H -H -CH,CHOHCH,OH -H -N(COCH3)CH2CHOHCH20CH3
f CCN -CH2CHOHCH20H -CH3 -CH2CHOHCH20H -H -NHCOCH20CH3
g CCCb -CH2CH20H -(R1) -CH2CH20H R -CON(CH2CH20H),
h CCCb -CH2CHOHCH20H -CH3 -CH2CHOHCH20H -H -CONH2
i CCN -CH2CHOHCH20H -H -CH2CHOHCH20H -H -N(COCH20H)CH2CH20H
j CCN -CH2CHOHCH20H -H -CH2CH20H -H -N(COCH3)CH2CHOHCH20H
k CCN -CH2CHOHCH20H -H -CH2CHOHCH20H -H -NCOCHOHCH2CH2CHZC
500 Radiopaques

Table 10.2 (Continued)

CONHCH3 CONHCH3

\
OCHN
HOCHzCHz

H OH OH OH
H OH OH OH
CHzOH CHzOH CHzOH
6 Organic Iodine Compounds

Table 10.2 (Continued)

COOH COOH
I I

a CNC -(Cl&J4- -CONHCH3

b CNH -(CH2), -H
c CNH -CH2(CH2OCH2),CH,- -H
d CNH -CH20CH2- -H
e CNH -(CHz)s- -CONHCH3
f CNN -CH2CH2S02CH2CH2- -N(C2H5)COCH3
g CNH -(CH20CH2), -H
h CNH -CH2(CH20CH2)3CH2- -H
i CNH -(CH,OCH,),- -H
COOH CONHCH3
I I

CHzOHwcHZN
OH OH H OH

(93(Ioxaglate meglumine)
5 02 Radiopaques

Table 10.2 (Continued)

COOH
COOH I

I NlI&o
OC-
H3C CH3 I H2C CH3
H3C
OC- I
1 NCH2 I H3C CH3
I I

(10)
(10) Type X R6 R1 R2 R3 R4
a CCN -COCH2CO- -CH2CH20H -CH(CH20H)2 -H -CH(CHzOH), -H
b CCN -CH2CHOHCH2- -COCH3 -CH2CHOHCH20H -H -CH2CHOHCH20H -H
c CCN -CO(CH2)2S(CHz)2CO- -H -CHzCHOHCH20H -CH3 -CH2CHOHCH20H -CH3
d CCN -COCH2CO- -CH3 -CH(CHOH)CH20H -H -CH(CHOH)CH20H -H
CH,OH CH,OH

C-N-X-N-C
I I

e CNC -CH,CHOHCH,- -H -CH(CH,OH), -H -COCH(OH)CH3 -H

COOH
I
6 Organic Iodine Compounds

Table 10.2 (Continued)

-
COOH
I

"L-Lactoyl residue;
ba 1,3,5-benzenetricarboxamideor trimesic acid amide;
c2-oxo-3-hydroxy-l-piperidinyl;
Table 10.3 Characteristics of Ionic and Nonionic Iodine-Containing Contrast Agents
Iodine Melting LD,,, g/kg Solubility, g/
USAN or INN Content Point or I (=g 100 mL H,O
Name Chemical Name Mol. wt. (%) ("c) Ikg) at 20°C PK, H.,O25 Ref.
A. Ionic monomers
Acetrizoate (2a) 3-Acetylamido-2,4,6-triiodoben- 9.56 94.20 2.08
[85-36-91" zoic acid
Sodium salt 5.51b Freely soluble
Meglumine salt 176
Diatrizoate (2b) 3,5-Bis(acetamin0)-2,4,6-triiodo- 11.0" 2.05
[737-31-51 benzoic acid
Sodium salt 11.4'; 8.41b 60 2.70 233
Meglumine salt 13.81' 89 272,273
Diprotrizoate (2c) 3,5-Dipropionamido-2,4,6-tri- 0.0118b
[129-57-71 iodobenzoic acid
Sodium salt - 50 233
Iodamide (2d) 3-(Acety1amino)-5-[(acetylami- logb 0.3 (22°C) 1.88 274,275
[440-58-41 no)methyll-2,4,64riiodoben-
zoic acid
Meglumine salt
Iotrizoic acid (2e) 2,4,6-Triiodo-3-[(I-0x0-
[16024-67-23 3,6,9,12,15-pentaoxa-hexadec-
1-y1)aminolbenzoicacid
Ioxotrizoic acid (20 3-(Acety1amino)-5-[(hydroxy-
119863-06-01 acety1)aminol-2,4,6-triiodo-
benzoic acid
3-(Acety1amino)-5-(acetylmeth-
y1amino)-2,4,64riiodobenzoic
acid
Sodium salt
Ioglicic acid (3a) 3-(Acety1amino)-2,4,6-triiodo-5-
L49755-67-11 [[[2-(methylamino)-2-oxoeth-
yllaminol carbonyl]benzoic
acid
Ioseric acid (3b)
[51876-99-41

nolbenzoic acid
Iothalamic acid 3-(Acetamino)-2,4,6-triiodo-5-
( 3 ~[2276-90-61
) [(methylamino)carbonyll-
benzoic acid
Sodium salt
Meglumine salt
Ioxitalamic acid 3-(Acetylamin0)-5[[(2-(hydroxy-
(3d)[28179-44- ethy1)aminol carbonyll-2,4,6-
41 triiodobenzoic acid
Iopanoic acid (4a) 3-Amino-alpha-ethyl-2,4,6-tri- 155.2-157 (d,l
[96-83-31 iodobenzene-propanoicacid form)
Iophenoxic acid Alpha-Ethyl-3-hydroxy-2,4,6- 143-144
(4b) 196-84-41 triiodobenzene-propionic acid
Ioprocemic acid 3-(Acetylethy1amino)-2,4,6-tri-
( 4 ~ L1456-52-61
) iodobenzene-propionicacid
Ipodic acid (4d) 3-[[(Dimethylamino)-methylenel
[5587-89-31 aminol-2,4,6-triiodobenzene-
propanoic acid
Sodium salt Freely sol.
Calcium salt 0.10
Tyropanoate (43) Alpha-Ethyl-2,4,6-triiodo-3-[(l- Sol.
[7246-21-11 oxobuty1)-aminolbenzene-pro-
panoic acid
Sodium salt
Iolidonic acid (40 Alpha-Ethyl-2,4,6-triiodo-342-
121766-53-01 oxo-l-pyrrolidiny1)ben-
zenepropionic acid
Iomorinic acid (4g) 2-Methyl-3-[2,4,6-triiodo-3-[I-(4-
[51934-76-03

acid
Iolixanic acid (4h) 2-[2-[3-(N-Ethy1acetamido)-2,4,6-
[22730-86-51 triiodo-phenoxylethoxylpropi-
onic acid
 5-LAcetyl(2-hydroxy-3-meth- 10.3g I/kg
oxypropy1)amino)-N,N' - (rats)
bis(2,3-dihydroxypropy1)-
2,4,6-triiodo-1,3-
benzenedicarboxamide
Iopromide (70 N,N'-Bis-(2,3-dihydroxypropy1)- 10.3 g Ikg
L73334-07-31 2,4,6-triiodo-5-(methoxy- (rats)
acety1)aminol-N-methyl-1,3-
benzenedicarboxamide
Iosimide (78) N,N,N1,N',N"N"-Hexakis(2-hy-
[79211-10-21 droxyethy1)-2,4,6,-triiodo-
1,3,5-benzenetricarboxamide
Iotriside (7h) ( 2)-N,N'-Bis(2,3-dihydroxy-pro-
[79211-34-01 py1)-2,4,6-triiodo-N-methyl-
1,3,5-benzenetricarboxamide
N,N'-Bis(2,3-dihydroxypropy1)- 186-198 18.3 g Ilkg
5-[(hydroxy-acetyl)(2-hy- (m.mice)
droxy-ethy1)amino-2,4, 18.1 g Ilkg
6-triiodo-1,3-benzene- (f.mice)
dicarboxamide
Ioxilan (5) 5-[Acetyl(2,3-dihydroxypropy)- 22.4 g I/kg
[107793-72-61 [amino]-N-(2,3-dihydroqpro- (m. rats)
py1)-N'-(2-hydroxyethy1)- 20.1g I/kg
2.4.6-triiodo-1.3-
, . (f.rats)
benzenedicarboxamide
5-[[Dihydro-5-(hydroxymethy1)-
2(3H)-furanylidenel-amino]-
N,N'-bis[(2-hydroxy)-1-(hy-
droxymethy1)-ethyl]-2,4,6-
triiodo-1,3-benzenedim-
boxamide
N-13-LAcetyl(2-hydroxyethy1)-
amino]-2,4,6-triiodo-5-[(meth-
ylamino)carbonyllphenyll-
D-gluconamide
Ioglucomide (8b) N,N'-[2,4,6-Triiodo-5-[(methyl-
[63941-74-21 amino)carbonyl]] 1,3-phenyl-
enelbis-D-gluconamide
Table 10.3 (Continued)
Iodine Melting LD,,, g/kg Solubility,g/
USAN or INN Content Point or I (=g 100 mL H20
Name Chemical Name Mol.wt. (%I (TI Ikg) at 20°C PK,H,O25 Ref.
Ioglunide (8c) N-[3-(Acetylmethy1amino)-5- 807.12
[56562-79-91 [[(2-hydroxy-ethy1)aminolcar-
bonyll-2',4',6'-triiodophenyl-
D-gluconamide
Iogulamide (8d) N,N'-Bis(2,3-dihydroxypropy1)- 881.16
L75751-89-21 5-[L-xylo-2-
hexulosonoyl)&hyphen
amino]-2,3,4-tri-iodo-
1,3-benzenedicarboxamide
Iosarcol(8e) 1-[[[[3,5-Bis(acety1amino)-2,4,6- 862.19
[97702-82-41 triiodobenzoyll-methylaminol-
acetyll-methylamino]l-deoxy-
D-glucitol
Metrizamide (80 2-[[3-(Acety1amino)-54acetyl- 789.10 230-240 (d) 14 Ib 17.5"
[31112-62-61 methylamino)-2,4,6-triiodo-
benzoyll-amino]-2-deoxy-D-
glucose
C.Ionic dimers (biscompounds)
Iocarmicacid (9a) 3,3'-[(1,6-dioxo-1,6-hexanediy1)- 1253.87
[10397-75-81 diiminolbis-[2,4,6-triiodo-5-
[(methylamino)carbonyll-
benzoic acidl
Meglumine salt 1644.31
Iodipamide (9b) 3,3'-[(1,6-Dioxo-1,6-hexanediy1)-1139.77
[606-17-71 diiminolbis-[2,4,6-triiodoben-
zoic acidl
Megluminesalt 1530.2 34" Sol. 2.76 (pK,) 320,321
Iodoxamic acid (9c) 3,3'-[(1,16-Dioxo-4,7,10,13-tet- 1287.93 52b 0.0364(20°C) 1.8 (pK,) 189,263
[31127-82-91 raoxahexadecane-1,16-diy1)-
diimino]bis[2,4,6-triiodoben-
zoic acidl
Meglumine salt 1678.36
Ioglycamic acid
(9d) [2618-25-91
triiodobenzoic acid]
Meglumine salt 281 (d) (three crys- Sol.
talline modifica-
tions)
Iosefamic acid (9e) 3,3'-[(1,10-Dioxo-1,lO-de- 1309.98 58.12 279 131b
[5591-33-31 canediy1)diiminolbis-[2,4,6-
triiodo-5-[(methylamino)car-
bonyll-benzoic acidl
Iosulamide (90 3,3'-[Sulfonylbis[(l-0x0-3,l-pro-
[63534-64-53 panediy1)-iminol]bis[(5-acetyl-
ethylmino)-2,4,6-triiodo-
benzoic acidl
Meglumine salt
Iotetric acid (9g) 3,3'-[(1,14-Dioxo-3,6,9,12-tet-
[60019-19-43 raoxatetra-decane-l,14-diy1)-
diiminolbis[2,4,6-triiodo-ben-
zoic acidl
Iotranic acid (9h) 3,3'-[Oxy-bis(ethy1eneoxy-ethyl-
l26887-04-71 enecarbony1imino)lbis-[2,4,6-
triiodobenzoic acid]
Iotroxic acid (9i) 3,3'-[Oxybis[2,1-ethanediyloxy-
[51022-74-31 (1-0x0-2,1-ethanediy1)iminoll-
bis[2,4,6-triiodobenzoicacidl
Ioxaglic acid (9j) 3-[M3-(Acetylmethy1amino)-
L59017-64-01 2,4,6-triiodo-5-[(methylamino)-
carbonyllbenzoyl]amino]
acetyllaminold-[[(2-hydroeth-
y1)aminol carbonyll-2,4,6-tri-
iodobenzoic acid
-
Meelumine salt
Sodium-meglumine salt
Iozomic acid (9k) 3,3'-[1,4-Butanediylbis[oxy(2-
[31598-07-91 hydroxy-3,l-propanediyl)(ace-
tylmino)llbis[5-(acetyl-
methyl-amino)-2,4,6-
triiodobenzoic acid
Table 10.3 (Continued)
Iodine Melting LD,,, gkg Solubility,gl
USAN or INN Content Point or I (=g 100 mL H,O
Name Chemical Name Mol.wt. (%) ("C) Vkg) at 20°C PK, H,O 25 Ref.
D. Nonionic bis compounds
Iodecol (lOa) 5,5'-[1,3-Dioxo-1,3-propanediy1)-
. . 1566.20 48.62 259
[81045-33-21 bis[2-hydroxy-ethy1)iminoll-
bis[N,N1-bis[2-hydroxy-1-(hy-
droxymethy1)ethyll-2,4,6-
triiodo-1,3-benzenedicarbox-
amidel
Iodixanol (lob) 5,5'-[(2-Hydroxy-1,3-propane- 1550.2 49.12 240-250
[92339-11-21 diyl)bis(acetyl-imino)l-
bis[N,Nf-bis(2,3-dihy-
droxypropy1)-2,4,6-triiodo-1,3-
benzenedicarboxamidel
5,5'-[Thiobis[(l-oxo-3,l-pro- 1608.35 47.34 240-260
panediyl)iminollbis[N,N'-
bis(2,3-dihydroxypropy1)-
2,4,6-triiodo-N,N1-dimethyl-
1,3-benzene-dicarboxamidel
Iotrolan (10d) 5,5'-[(1,3-Dioxo-1,3-propanediy1)-1626.25 46.82 26 gPlrg
c79770-24-41 bis(methy1-imino)lbis[N,N'- (mice)
bis[2,3-dihydroxy-1-(hydroxy- 12.7g k g
ethy1)propyll-2,4,6-triiodo-1,3- (rats)
benzene-dicarboxamidel
N,N'-(2-Hydroxy-1,3-pro-
panediyl)bis[N1-[2-hydroxy-1-
(hydromethy1)ethyll-5-[(2-
hydroxy-1-oxopropyl)aminol-
2,4,6-triiodo-1,3-benzenedi-
carboxamidel,[S-(R*&*)I
E.Miscellaneous
Ioxabrolic acid (11) N-(2-Hydroxyethy1)-2,4,6,-tri- 1127.88 Br:
[96191-65-01 iodo-5-[2-[2,4,6-tribromo-3- 21.25
(N-methylacetamido)-5- I: 33.75
(methyl-carbamoy1)benzi-
amidolacetylamino-l,3-ben-
zenecarboxamidel
 Tris(iotha1amic 5,5',5-(Nitrilotriacety1triimino)-
acid) (12) tris (2,4,6-triiodo-N-methyl-
isophthalamic acid)
Iodoalphionic acid 3-(4-Hydroxy-3,5-diiodopheny1)-
(13)[577-91-31 2-phenyl-propionic acid
Disodium salt >230
Iophendylate (14) Ethyl 10-@-iodophenyl)undecy- Bp: 196-198 Slightly sol.
[99-79-61 late
Iopydone (15a) 3,5-Diiodo-4(lH)pyridinone Insol.
[5579-93-11
Iodopyracet (15b) 3,5-Diiodo-4-0~0-(4H)-pyridine 6.3b;3.2 Ib 2.15
[300-37-81 acetic acid
2,2'-Iminodiethanol(1:l)salt 2 (i.v. in 60
dogs)
Iopydol(15c) 1-(2,3-Dihydroxypropy1)-3,5-
[5579-92-01 diiodo-4(lH)-pyridinone
Propylidone (15d) 3,5-Diiodo-4-0x0-1-(4H)-pyr- 0.3b 0.014 (15")
tn [587-61-11 idineacetic acid, propyl ester
4
-L Sodium io- 1,4-Dihydro-3,5-diiodo-1-methyl- 4.6; 2 Ib Freely sol.
domethamate 4-0x0-2,6- pyridinedicarboxy-
(Iodoxyl) (154 lic acid, disodium salt
[519-26-61
Iopax 5-Iod0-2-oxo-l(2H)-pyridineace- 8' Very sol.
tic acid, sodium salt
"CAS Registry Number;
bi.v.in mice;
1.v. in rats;
C'

doralin mice;
"oral in rats.
51 2
their Chemical Abstracts Service (CAS) Regis-
try Numbers are listed in square brackets be-
neath each name.
6.2 Synthesis
The synthesis of iodinated ionic and nonionic
contrast agents follows a general pattern,
which includes (1)the selection and synthesis
of an intermediate containing the ring nu-
cleus, (2)introduction of an activating group,
(3) iodination, and (4) acylation.
The compound usually selected as the
starting material is a substituted benzoic,
isophthalic, isophthalamic acid, or one of their
derivatives (227). The activating group is in-
troduced as a nitro group followed by reduc-
tion to an amino group before iodination. Re-
duction of the nitro group is effected by
catalytic reduction using 5-10% palladium on
charcoal or Raney nickel as catalyst (228,229).
The latter gives a higher yield and a cleaner
product than those of other reduction meth-
ods. Ammonium or sodium sulfide may also be
used as a reductant (186, 230-232). The
amino group can also be diazotized to form a
phenolic hydroxyl group.
A vinyl C-I bond is more stable than an
alkyl C-I bond. The phenyl ring, in the pres-
ence of an activating group, can accept three
iodine atoms upon iodination with iodine
monochloride (233). Iodination is usually car-
ried out with iodine monochloride in dilute hy-
drochloric acid or dilute acetic acid or in potas-
sium chloride solution (180, 233, 234).
Acetylation of the amino group changes the
electron density of the ring and locks the car-
bon-iodine bond in place (201, 227). The sub-
stitution also increases the solubility and re-
duces the toxicity of the iodinated product.
Acetylation may be effected by using ketene in
the presence of sulfuric acid (232). A general
method of acylation is the dissolution of the
amine in dimethylformamide or dimethylacet-
amide (235),followed by addition of the appro-
priate acyl chloride.
More synthetic steps and more different in-
termediates are required to complete the syn-
thesis of nonionic contrast agents than that of
ionic contrast agents. The commonly used in-
termediates for introducing (po1y)hydroxyal-
kyl groups to the triiodophenyl ring in a
nonionic contrast molecule are amino-alco-
Radiopaques
hols, epihalohydrins, 1,3-dihydroxy-2-amin-
opropane (serinal) (236), 3-(N-2-hydroxy-eth-
y1)amino-1,2-propanediol(2371, and so forth.
Other reagents, such as 1,1,2-trichloroethane
(238) and acetoxyacetyl chloride (238), may
also be needed for the synthesis. Acetoxyacetyl
chloride is a protective reagent for the hy-
droxyl groups. Any of the intermediates re-
quired for the synthesis of nonionic contrast
agents would have to be prepared, purified,
and recovered for reuse if necessary. Felder
(196) remarked on the complexity of the prep-
aration of iopamidol that "Methods of analysis
were developed with specifications for 35 raw
materials, five intermediate products, and the
final product, for a total of 289 single analyti-
cal tests."
In the following sections, selected examples
of synthesis for ionic and nonionic, and mono-
meric and dimeric contrast agents are given to
illustrate the general synthetic approach. The
synthesis of an ionic monomer iocetamic acid
(6a),a triiodoanilide of the NNH type, used for
oral cholecystography (240),is shown in Equa-
tion 10.4.
The synthesis of an ionic contrast agent is
almost complete at this stage. The product is
crystalline and can be readily isolated. The
crude product can be purified by precipitation
from a salt solution with acid or by recrygtal-
lization from solvent (201). In the synthesis of
nonionic contrast agents, additional synthetic
steps are required to attach the (po1y)hydroxy-
alkyl substituent groups to the ring. The pres-
ence of these groups in the molecule increases
reactivity and water solubility and also makes
the product isolation from an aqueous solu-
tiondifficult. If the product is amorphous and
cannot be crystallized, its isolation may re-
quire flash evaporation or vacuum distillation.
To concentrate and purify iopamidol from
aqueous solution, permeable filtration mem-
branes are used in special filtration proce-
dures (241). Also, electrolytes introduced in
the intermediate synthesis should be removed
by desalination or ion exchange. Deionization
and purification of the radiopaque can be
achieved by column chromatography by use of
silanized silica gel for adsorption, followed by
washing with water until free of electrolyte,
and desorption with dilute organic solvent
(242). For example, crude ioversol can be ei-
6 Organic Iodine Compounds
Iocetamic acid
ther purified by reversed-phase liquid chroma-
tography (243) or purified continuously with a
bed of mixed ion-exchange resin contained by
selectively permeable membranes with an
electric potential imposed across the mem-
brane (244). Recrystallization from a butanol
mixture is another way of purifying iopamidol
to pharmacopoeia standard (245).
One of the methods for synthesis of iohexol
(7b),a nonionic contrast agent, is shown in
equation (10.5) (246). Nonionic contrast me-
dia are more costly to manufacture than are
ionic constrast media. The cost of nonionic
contrast media is determined by the number
of synthetic steps, overall yield, cost of the in-
termediates, difficulties in isolation and puri-
fication, and other associated manipulations.
Many alternate paths of synthesis and purifi-
cation of nonionic contrast agents have been
reported. For example, the nonionic contrast
agent ioxilan (7j) can alternately be prepared
from the ionic contrast agent ioxitalamic acid
(3d) by reaction with epihalohydrin, to selec-
tively produce the N-hydroxyalkylated prod-
uct (202, 247). Iohexol can be prepared from
the triacetyl derivative of 5-amino-2,4,6-tri-
iodoisophthaldiamide by N-allylation with al-
lyl chloride or allylamine, followed by oxida-
tion and hydrolysis (i.e., deprotection), to give
the product (248). The cost of manufacture
can be reduced by using a minimal excess of an
expensive reagent (acetoxyacetyl chloride) or
by shortening the purification process (239).
Selective hydrolysis of the protected product
in ioversol synthesis can simplify the purifica-
tion procedures (249). Methods for iodine re-
covery from manufacturing wastes of iodin-
ated contrast media may also contribute to the
reduction of the manufacturing cost (250,251).
Novel reactions can also be used to prepare
nonionic contrast media (252). For example,
iomeprol in alkaline solution undergoes the

COOH
H2N
I
QOOH
I
Ac20
AczN
lI$:ocl
H2NCH2CHC(OH)H20H
(Et3N in DMF) *
AczN
AczN
T I
Epichlorohydrin
~N~HCO~ICH~OH:H,O)
(Refluxing)
AcNI CONHCHzCHOHCHzOH
CH2 I
I
CHOH Iohexol
Smiles rearrangement, an intramolcular nu-
cleophilic aromatic substitution, in which the
substituent 5-[(hydroxyacetyl)methylaminol
group is rearranged to form the 54methylami-
nocarbonylmethoxy] group. This reaction un-
couples and replaces the N--C bond with the
0-C bond between the substituent and the
ring intramolecularly (253). The rearrange-
ment is reversible, with sodium hydroxide or
sodium methoxide catalyzing the forward re-
action. Ioversol, iopamidol, and a dimer also
undergo the Smiles rearrangement. The reac-
tion is useful for preparing nonionic iodinated
radiopaques that cannot be obtained by con-
ventional synthetic methods, such as by con-
verting from a CCO type to a CCN type molecule
(252).
Many side reactions can occur during the
alkylation of an intermediate containing
many nucleophilic sites. Bjoersvich et al. (254)
studied the influence of various cations on the
N/O regioselectivity in the N-alkylation of
acetamidotriiodoisophthalamide derivatives
with 3-chloro-1-methoxy-2-propanol and found
that the Ca2+ion gives the best selectivity and
the highest yield of the desired nonionic prod-
uct. A recent patent showed that the presence
Radiopaques
COOAc
I
COOAc
- SOCl2
CONHCH2CHOHCH20H
CONHCH2CHOHCH20H
(10.5)
AcHN CONHCH2CHOHCH20H
of Ca(OH), can selectively influence the hy-
drolysis ofthe protected groups (255).
Hexaiodinated bis compounds are formed
using an alkylene bridge or a substituted alky-
lene bridge through coupler groups, such as
amide (-40-NH-) or reversed amide
(-NH--C&) or mixed amide and reversed
amide, to link two triiodinated monomers.
The bis compounds with amide-linked bridges
are synthesized by condensing two molecules
of a triiodinated monomer containing a free
amino group with one molecule of a dicarboxy-
lic acid dichloride (189, 235, 256). The bis
compounds with reversed amide groups are
prepared by condensing two molecules of sub-
stituted triiodinated isophthalic acid mono-
chloride with an (un)substituted alkylene dia-
mine (201). Bis compounds (e.g., ioxaglate)
containing mixed linkages with one amide and
one reversed amide group in the molecule are
asymmetric (199, 257), whose synthesis is
-
more involved than that of com~ounds with
symmetric anchoring groups. The synthesis of
an amide-linked ionic bis compound iocarmic
acid (9a)is shown in Equation 10.6.
The synthesis of nonionic bis compounds is
analogous to that of ionic compounds, except
6 Organic Iodine Compounds
2 COOH
I
COOH
Iocarmic acid
that provisions should be made for the subse-
quent introduction of the polyhydroxyalkyl
groups (258). An example taken from the
patent literature (259) is shown in Equation
10.7.
6.2.1 Isomerism. Iodinated radiopaque com-
-pounds that contain a chiral center or chiral
centers form optical isomers or diastereoiso-
mers. The presence of an asymmetric carbon
atom in iopanoic acid (220),iophenoxic acid, or
iodoalphionic acid (225), for example, leads to
the formation of d and I forms. In iocetamic
acid, free rotation around the axis connecting
the iodinated phenyl nucleus with the tertiary
nitrogen atom in an alkylamido substituent is
impeded by the bulky ortho-substituted iodine
atoms, creating d and I forms (192). The mol-
ecule also contains an asymmetric carbon
atom that gives rise to d' and I' forms. The
racemates contain d,dr and 1,l' as well as d,l'
and dl,lforms, which are mutually diastereoi-
someric and have different melting points and
solubilities. Mixtures of diastereoisomers ex-
hibit a wide melting range over many degrees.
Besides diastereoisomerism, iodinated ra-
diopaques may show polymorphism; crystals
formed under different conditions show iden-
tical infrared absorption bands but with dif-
ferent intensities (262,263).
Nonionic contrast media have a multi~lic-
A
ity of optical and rotational isomers and dia-
stereoisomers (201, 261, 264-267). Optical
isomerism is derived from the chiral centers in
COOH
polyhydroxyalkyl groups, and geometric iso-
mers from the restricted rotation of the pen-
dant substituents attributed to steric hin-
drance about (1)the amide (CO-N) bond, (2)
the aryl-nitrogen bond in N-substituted acet-
anilides, and (3) the aryl-carbon bond in ben-
zamides (266). Nonionic contrast agents, such
as iopamidol, iohexol, iopentol, ioversol, iox-
ilan, and iotrolan, exist in aqueous solution as
mixtures of isomers. Iohexol exists in two iso-
mers in a ratio of 1:3 (endolexo) separable by
HPLC, and the exo-iohexol has a longer reten- .
tion time than that of the endo-iohexol (267).
Iopentol exists in several diasteromers that
may not be readily resolvable and identifiable
by available analytical techniques (266). Io-
verso1 has four apparent rotamers that can be
resolved by reversed-phase HPLC chromatog-
raphy and confirmed as acetonide derivatives
by high resolution NMR spectroscopy (264).
HPLC and NMR spectroscopy show that the
rotational isomers of iohexol and of ioxilan are
interconvertible after equilibration (265).
The rotational isomers exist in cis and
trans, anti and syn, or endo and exo forms. The
cis and trans forms arise from slow rotation of
the side-chain carbonyl groups, hindered by
adjacent ortho-substituted iodine atoms in the
ring, resulting in the two carbonyl groups
aligned either parallel or antiparallel to each
other (268,269). The anti and syn forms refer
to the position of N-methyl or N-alkyl group
relative to that of adjacent carbonyl oxygen
(266). The endo and exo forms refer to the po-
516
ClOC
+ ClCOCH2COCl - ClOC
I#
I I
sition of the arnide group or the N-acetyl's
methyl group with reference to the plane of
triiodophenyl ring. The various geometric iso-
mers will coalesce into one form at higher tem-
perature (265). The dimer or bis compound
iodixanol has three geometric isomers: exo-
exo, exo-endo,and endo-endo forms that can be
adjusted to elute as a single peak by HPLC for
the purpose of quantitation (270).A multiplic-
ity of isomeric forms in nonionic contrast
agents is necessary for high water solubility
because it increases the energy barrier of de-
solvation and slows down the process of crys-
tallization and precipitation. From the view-
point of drug design, a higher water solubility
minimizes the binding to biomacromolecules,
decreasing the toxicity and increasing the bio-
Radiopaques
NHCOCHzCHONH
I
/
logical tolerance. It is an attribute that should
be custom designed into any improved non-
ionic contrast molecules.
6.2.2 Potential Radiopaques. A large num-
ber of organic iodine compounds synthesized
in search of improved contrast agents are de-
scribed in the patent literature; limitation of
space does not allow a full tabulation of these
compounds. Because information concerning
them may prove to be of value in the design
and synthesis of new potential contrast
agents, this chapter lists only the potential
nonionic radiopaques, where the main current
research effort is centered. Summaries of re-
search on ionic contrast media may be found
in early references (211,271).
6 Organic Iodine Compounds
Table 10.4 lists potential nonionic contrast
agents containing one triiodinated 1,3-ben-
zenedicarboxamide nucleus and variations.
Table 10.5 lists hexaiodinated nonionic com-
pounds containing two such rings per mole-
cule. Tables 10.6-10.9 contain examples of
polymers and other derivatives that have a dif-
ferent iodine-carrying moiety. Physicochemi-
cal information, toxicity, and proposed use are
given when available. Contrast media are used
in large quantities in medical diagnostic radi-
ology. The nonionic agents generally surpass
ionic ones in offering higher acceptance and
safety to the patients.
6.3 Structure-Activity Relationship
With few exceptions, organic iodine com-
pounds for X-ray diagnostic use contain an io-
dinated benzene ring or an iodinated pyridone
nucleus. For reasons of low toxicity and high
biotolerance, modern contrast agents are vari-
ations of structures based on the triiodophe-
nyl moiety as the iodine carrier. According to
Hoey and Smith (180,201),the desirable prop-
erties of intravascular contrast agents should
include the followine:
"
Opacity to X-rays.
High water solubility at high iodine
content.
Chemical stability.
Biological safety.
Intravascular contrast agents should have
low viscosity and low osmolality as close as
possible to those of body fluids, to improve tol-
erance and minimize adverse reactions. Con-
trast agents intended for oral cholecystogra-
phy should possess hydrophilic and lipophilic
properties, so that the compound can be orally
absorbed, should be excreted from the liver
and bile ducts in sufficient amount to provide
radiopacity, and should have no adverse side
effects. Because each of these properties is as-
sociated with certain molecular features and
not all of them are structurally compatible,
the best radiopaques represent a compromise
of a maximum of these desirable qualities.
In the following sections, relations between
structure-dependent properties and charac-
teristics of radiopaques are given.
517
6.3.1 Substituent Structures. The basic struc-
ture in an iodinated contrast agent, the triio-
dinated phenyl ring, is lipophilic. The lipophi-
licity can be partially or completely modified
by the substituent groups, shown as R, X, and
Y in (1).The carboxylic acid moiety in the R
group is responsible for the water solubility
and ionic property of ionic contrast media. The
X and Y substituent groups, containing
acylamino, substituted acetamido, substituted
carbamoyl, 0x0, carbonyl, hydroxy, methoxy,
or ether linkages, determine or modify the
physicochemical properties (such as water sol-
ubility and hydrophilicity) and the pharmaco-
logical properties (such as biotolerance, toxic-
ity, distribution, excretion of the molecule).
For example, N-alkylation increases the dis-
tribution of the contrast agent in the liver as
compared to the corresponding nonalkylated
compound (263,400). Asymmetry in substitu-
tion, such as replacement of an acetamido
group with an N-methylcarbamoyl group, can
increase the water solubility of iothalamate
compared to that of diatrizoate (9). Many ionic
contrast molecules show distinct hydrophilic
-
and lipophilic regions, and only a few have hy-
droxyl
- groups.
- - The nonionic contrast mole-
cules have a large number of hydroxyl groups,
masking the lipophilic regions of the molecule
to achieve high water solubility. ..
The ionic charge of the carboxyl group
leads to the high osmolality of ionic contrast
media and is the cause of neurotoxicity and
vascular pain in arteriography (181,182). Me-
trizamide, the first-generation
- nonionic con-
trast agent, induced no adverse reactions
when first introduced in X-ray diagnosis. This
confirmed AlmBn's theory (181)that nonionic
radiopaques are safer and better tolerated
than ionic ones and led to the rapid develop-
ment of nonionic radiopaques in the 1980s,
which has continued to the present. The use of
nonionic contrast media has been beneficial to
high risk patients. The compounds listed in
Table 10.3 are second-generation monomeric
and dimeric contrast media, consisting mainly
of derivatives of 5-amino-1,3-benzenedicar-
boxamide or 5-amino-isophthalamide. These
compounds have no ionizable carboxyl groups
and rely on the presence of 4-6 hydroxyl
groups per monomer molecule or 8-12 hy-
droxyl groups per dimer or bis compound mol-
518 Radiopaques

Table 10.4 Potential Nonionic Monomeric Radiopaques

h d .
e ISubclassl
. - -.- Stem Structure Comments Ref.
1. CCN type

R alkyl, CH,Ph, CH,0COCH3, CH,OCOC(CH,),, or

= Synthesis
CH,CCl,; R1 = COCH,.
R = C1-12 alkyl, R1 = CH2CH20H Synthesis
COzR
I

R = phthalidyl, C, alkyl, CH,Ph, CH(CH,)OAc, Synthesis (13 esters prepared) 344

CH(CH,)O2CC(CH3),, CH20,CC(CH,),, or
CH,CH,N(CH3),;
R = N(CH,)COCH,, CONHCH,

Synthesis; bronchography, 345

hepatography.

R = CH,C02H or CH,CONH,; R1 = OH or NH Synthesis, toxicity studies . 346

CH3 Urography, arteriography,
myelography
6 Organic Iodine Compounds 519

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.
CONHR
I

I
R = CH,, CH,CH,OH; R1 = CONHMe or NMeAc; R2 Synthesis
3
= gluconoyl; R = H or Me.

1
R = H, Me, Et, Pr, CHMe,, CH,, C H S H , , Synthesis (60 compounds
(CH,),OMe, Ph, CH,Ph; R1 = Me, Et, prepared)
CH,CH=CH,, or QCO,H, Q = CH,CHMe; R2 = H,
Me, Et, CH,CO,H, or (CH,),CO,H; R3 = H, Me, or
Et; R4 = Me, Et, or Ph; R5 = H, C02H, or CONHMe

R = H; R1 = H, Me, or CH,CH,OH; NRR1 = Synthesis

morpholino; X = amino acid residue; R2 = Me,
CH,OMe, or CH,OH
CHzX
I

I
X = OH or C1; R = NH,, NHAc, NAc, or I; R1 = NH,, Synthesis (11compounds 351
NHAc, NAc,, NHCOPr, OMe, or I. prepared)
520 Radiopaques

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.

Q = chemical bond, (CH,),, or OCHEt; R = OH or C1; Synthesis (11compounds 351

2
R1 = NH,, NHAc, NAc,, or I; R = NH,, NHAc, prepared)
NAc,, NHCOPr, OMe, or I.
Q = chemical bond, (CH,),, or OCHEt; R = CH,, Synthesis 352
CCl,, Bu, CH,CO,Me, or CH,CMe,COMe; R1 = H,
NH,, or NHAc; R2 = H or CONHMe.
COX

I
X, Y = OH or NRR1 with R = H, Me, or CH2CHOH; Synthesis
R1 = H or Me; NRR1 = morpholino; (A = residue of
an amino acid, e.g., glycine, DL-serine, DL-alanine,
sarcosine, proline, or glycyl-L-leucine;Z = H or
COR, with R = Ac, COCH,OMe, COBu, or
COCH,OH

R1 = (hydroxyalky1)amino;R2 = amino; R3 = alkyl, Dimers prepared from I

hydroxymethyl, ethoxymethyl, etc.; R4 = H, Me,
alkoxymethyl, etc.; R3R4 = alkylene
COR
6 Organic Iodine Compounds 521

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.
COR1
I

I I I
R4 I
I: R1 = N(CH2CH20H)CH2CHOHCH,0H; R2 = e.g., At least one amide group is 237
R1, NHMe, NHCH2CH20H;R3 = e.g., Me, CH20H, derived from amino-alc.,
CHMeOH; R4 = e.g., H, Me, CH2CH20H;or R3 and 3-(N-2-hydroxy-ethyl)-
R4 together from group I1 (n = 0, 1, 2,3; q = 0, 1,2, amino-1,2-propanediol.
3. Methods of preparation
given.
CONHR

I
R = CH(CH20H),, CH2CHOHCH20H;R1 = Ac, R2 =
CH2CHOHCH20Me.

R1 = H, alkyl, hydroxyalkyl; R2 = (p01y)hydroxyalkyl;

R3 = alkyl, hydroxyalkyl, alkoxyalkyl; R4 = H, alkyl,
( p l y)hydroxyalkyl.
CONR2R3

I: R1 = NR6COR5, CONR7R8; R2, R7 = CI4 (p01y)- Preparation of I1 is given. 358

hydroxyalkyl; R3, R8 = H, C14alkyl; R4, R6 = H,
C14 (hydroxy)alkyl, C14 (po1y)hydroxy-alkyl; R5 =
C14 (hydroxy)alkyl, C1, (po1y)hydroxyalkyl.
522 Radiopaques

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.

R = Ac, R1= H A variation of process for 359

preparation of ioversol.

I: R = H, OH; R1 = 1,3-dihyroxypropyl, 2,3- Ca(OH), used to promote 255

dihydroxypropyl. 11: X = acyl, Y = H, acyloxy; R1 = yield of main product.
1,3-dihydroxypropyl, 2,3-dihydroxypropyl.
OH
I
CONHCH2CHCH20H

II
O
X-C-N CONHCH2CHCH20H
I I
CH2 I OH
I
CH2
I
Y I
I: X = CH,OH, CH,OCH,; Y = OH, OMe. Two examples given with 360
LD,, values of 17.3 and
16.4 g 1/kgin mice.

R = H (I), CH,CH,OH (111, CH,CHOHCH,OMe (111) I1a t 350 mg I/mL has 361
osmolality 735 mOskg and
viscosity 9.4 CPat 37"C,
LD,, 1.460 mg Vkg in rat.
Better than iohexol.
6 Organic Iodine Compounds

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.
COR

cH3
11: R = -C1, -NHCH2CH,0H Intermediates for synthesis of 362
nonionic contrast agents

Synthesis of ioversol
derivatives and testing of
the safety of subsituent
group
11: R1 = H; R2 = CH3; R3 = H, CH3; R4 = H, Synthesis 363
CH2CH20H.
111: R1 = H; R2 = CH2CH20H;R3 = CH3, CH20H, Synthesis 363 .
CH(CH3)OH,CH(OH)CH20H,CH20CH3;R4 = H;
R3R4 = -CH(OH)CH2CH2CH2-.
IV: R1 = H; R2 = CH2CH(OH)CH20H;R3 = CH3, Synthesis 363
CH20H, CH(CH3)OH,R4 = H.
V: R1 = CH2CH,0H; R2 = CH2CH(OH)CH20H;R3 = Synthesis 363
CH3, CH20H, CH(CH3)OH;R4 = H.

R6
OH
I
2
I: R1, R = CH2CH(OH)CH20H,CH(CH20H)2, Synthesis
CH2CH20H,etc.; R3, R4 = H, Me, CH2CH20H;R,,
R6 = H, alkyl, CH,CH,OH, CH,OH, OH; Y = bond,
CH2CH2,CH20, OCH,, NCH,, CH2NCH2,0 , N;
m = 0,l.
II: Method of preparation given 364
524 Radiopaques

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.

(A), (B),(C), (D), (E): R, R1 = -H, -R, -OH, -OR, Synthesis (This group of 313,314
-hydroxyalkyl, -alkoxyalkyl; X1 = =N(CO)H, compounds with a
=N(CO)-alkyl, -S-, or - 0-; X2 = X1 or --CHZ-; m = heterocyclic ring has an
1-4,or if X2 is--CH2-,m = 0 4 ; n = 0-3; p = 2-5; q, r unexpected degree of
= 1 or 2 independently; and s = 2-5. anticoagulant behavior.)

R = CHzCHOHCH20H, Synthesis and testing

CH(CH20H),; R1 = H; R2 =
H, Ac, R, = H,
CH,CHOHCH,OH,
CHzCHOHCH20Me,
CHzCH(OMe)CH20H.

Xz
X,, X, = -H or alkoxy groups of 1-6 carbon atoms Synthesis; intravascular and 366
CNS uses
6 Organic Iodine Compounds 525

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.
2. The CNN type
CONRR~

I
I
NRR1 = glucosimino or N-methylglucosimino, R2 = H, Synthesis, angiography, 367
Ac, R3 = Ac, R4 = H, Me, R5= Ac. myelography
COR1
I
I

AcNH

I
R = H, Me; R1 = NHCH2CH20H I (R = H) has a solubility of 368
>SO% (w/v) in H 2 0 at 20°C
and a toxicity of >16 g k g
i.v. in rats.

Preparation and formulation 369

I, 11: Ac = acetyl, propionyl, 2-hydroxyacetyl, 2- Analogs with n = 4 have 370

methoxyacetyl or 2-hydroxy propionyl; R1, R2 = lower osmolality and higher
methyl, ethyl, propyl, butyl, 2-hydroqethyl, 2,3- viscosity than analogs with
dihydroxypropyl, 1,3-dihydroxyisopropyl; R3 = n = 2.
hydroxymethyl, 1,2-dihydroxyethyl, 1,2,3-
trihydroxypropyl, 2-hydroxyethoxy, 2,3-dihydroxy-
propoxy; n = 2,3,4.
526 Radiopaques

Table 10.4 (Continued)

-- ~~

Type (Subclass) Stem Structure Comments Ref.

CONR~R~
I

I
CHCHzOH
I I
CHzOH
I: R2 = H, C1, (po1y)hydorxyalkyl; R3 = H, C14 alkyl; An example prepared in 3 371
R4 = H, alkyl or (po1y)hydroxyalkyl; R1 = steps and purified by HPLC
NR4COCH(CH20H),, NR6COR5; R5 = C1* alkyl or is given.
(po1y)hydroxyalkyl; R6 = groups cited for R4,
CONR7R8; R7 = C14 (po1y)-hydroxyalkyl;R8 = H,
C,, alkyl.

3. The CNO type

R = P-D-glucopyranosyl, P-D-2,3,4,6-tetra Synthesis

acetylglucopyranosyl

4. The NO0 type

0

R, = CH3, CH,OH, CH(MEOH(1)-, CH,0CH3, Synthesis and testing 372

arabinos-2-yl; R = glucos-3-yl, allos-3-yl, fructos-3-yl
/ 6 Organic Iodine Compounds 527

Table 10.4 (Continued)

Type (Subclass) Stem Structure Comments Ref.
5. Mixed brominated and
iodinated CCN and CNN type
COY

I I
X
X = Br, iodo; Acyl = C2-, hydroxyalkanoyl, Synthesis
alkoxyalkanoyl, alkoxyhydroxyalkanoyl,
(un)substituted C, alkanoyl; R = H, C,-, alkyl,
hydroxyalkyl, alkoxyalkyl, alkoxyhydroxyalkyl,
H(OCH,CH2),. Me(OCH,CH,),, Et(OCH2CH2),,
alkylene analog of I; Y = HO, alkoxy, hydroxyalkoxy,
alkylamino, etc.; Z = COY,
hydroxyalkylaminocarbonyl, C, acylamino,
hydroxyacylamino, N-alkylacylamino, N-
hydroxyalkylacylamino, acylaminomethyl.

R O y C O N R 3 R 4
X I
R = hydroxyalkyl, (poly)alkoxyalkyl,polyhydroalkyl, Synthesis
aminocarbonylalkyl, etc.; R1, R3 = H, alkyl,
hydroxyalkyl; R2, R4 = hydroxyalkyl; X = iodo, Br.

(Only one compound prepared) Synthesis

6. Tetraiodinated CCI type

&
R ~ R ~ N

0 I 0
~ ~ 1 . 2

I HO
0
0
N
H
O
{~
OH

OH

OH
I: R1, R3 = H, alkyl, etc., R2, R4 = nonionic Synthesis 376
hydrophilic group; 11: (preparation given) I1
528 Radiopaques

Table 10.5 Potential Dimeric Radiopaques: Bis(2,4,6-triiodophenyl)Derivatives

Type (Subclass) Stem Structure Comments Ref.
1. CNN type (iodobenzamides)
NR(CH2)nR1 NR(CH~),R'
I I

I I
R = (un)substituted acyl; R1 = hydroxylalkyl, High water solubility, low 370
hydroxyalkoxy, etc.; R2, R3 = H, alkyl, hydroxyalkyl, osmolality, low viscosity
etc.; Z = bond, alkylene, etc.; n = 2 4 .

2. CCN type (iodoanilides)

R, R', R1, R1' = same or different = H, C,, alkyl, Methods of preparation given 377
linear or branched C1-4 mono- or poyhydroxyalkyl;
R ~R2',
, R3, R3' = same or different = CH2CH20H,
CH2CHOHCH20H,CH(CH20H)CHOHCH20H,
CH2(CHOH),CH20H, CH(CH,OH),; X = CH(OH),
CH(CH20H),C(OH)(CH20H),C(CH20H)2,
enantiomers, diastereoisomers, and/or rotamers.

3. CCN type (iodoanilides)

.

X = (CH,),, CH20CH2CH20CH,, CH,CH2SCH2CH2, 19 compounds prepared

CH2(OCH2CH2),0CH2R1 = CH(C0NHMe)
CH20Me, CHMeCONHMe, CH,CONHMe, Me, H;
R2 = H, Me; R3 = H, Me; R4 = H, CHMeC02H,
CH2C02H
CONR~R~ CONR~R~
I I

R1 = mono- or polyhydroxyalkyl; R2 = H, alkyl, R1; Methods of preparation given

R3 = mono- or dihydroxyalkyl; Z = bond, alkylene
optionally with 0 interrupters or OH or alkoxy
substituents.
1
I
/ 6 Organic Iodine Compounds 529

I
Table 10.5 (Continued)
; Type (Subclass) Stem Structure Comments Ref.
4. CCN type (iodoanilides)

R1 = H, alkyl, R2; R4 = (po1y)hydroqalkyl; R3 = alkyl, Methods of preparation given 378

R4; R5 = H, alkyl, poly(hydroxyalky1);X = (HO- or
alkoq-substituted)(O-interrupted)Cl-, alkylene.

5. CCN type (iodoanilides)

I: R1 = H, lower alkyl, R2; R2 = straight or branched Methods of preparation given 379

mono- or polyhydroxyalkyl; R3, R4 = H, lower alkyl;
n = 1, 2.

11: R, R1 = same or different, a straight or branched 28 compounds and methods of 380

mono- or poly-hydroqalkyl Cl-C3 residue preparation given
containing 1 to 2 OH groups; R,, &, R3 = H or CH3;
R,', R,', R3' = H, or CH,; Alkyl =
-CH(CH20H)CH(OH)CHzOH,-CH(CH20H)2,
-CH2CH-(OH)CH20H,-CH2(CHOH)4CH20H, or
-CH2CH20H; X = -CH(OH)-, -CH((CH20H)-
-CH(OH)(CH,OH)- or -C(CH,OH),-.
530 Radiopaques

Table 10.5 (Continued)

Type (Subclass) Stem Structure Comments Ref.
6. CNC type (iodobenzamides)
CONR~R~

R = straight or branched mono- or polyhydroxyalkyl Methods of preparation given 380

Cl-C3 residue containing 1 to 2 OH groups; R1, R2,
R3 = H, Me; R4 = HOCH2CHCH(OH)CH20H,
CH(CH2,CH2CH(OH)CH20H,
CH2(CHOH),CH20H or CH2CH20H;Z = CH(OH),
CH(CH,OH), CH(OH)CH,OH, or C(CH20H),).

7. CCN type
(iodobenzenedicarboxamides)
CONR~R~

R1, R4 = CZ-4polyhdroxyalkyl; R2 = H, Me, 42 compounds prepared

polyhydroxyalkyl, CH2CH20Me,
CH2CH(OH)CH20Me;R3, R8 = H, Me, CH2CH,0H;
R5 = Me, CH20H, CH20Me; R6, R7 = H, Me,
CH2CH20H,CH2CH20Me,CH2CHOHCH20H,
CH2CHOHCH20Me;or R5R2 = CH2CH2CH2
bearing 0-3 OH groups.
11: R1= R4 = CH2CHOHCH20H,Rz = R3 = R8 = H, LD,,: 25-28 g/kg (rats); 152 381,382
R5 = HOCH,, R6 = R, = Me. mOsm/kg at 300 mg IImL;
7.0 mPa, 52.1%I;
comparable to iotralan.

8. CCN type
(iodobenzenedicarboxamides)

R 2 0C N-C- (CH2)hC-N
I II
I R5 0 I I1
R2 = R1,
11: R1 = N(CH2CH20H)CH2CHOHCH,0H; Methods of preparation given 237
5
NHMe, NHCH2CHOHCH20H;R = H, Me,
CH2CH20H, CH2CHOHCH20H,CH20Me, m =
04.
6 Organic iodine Compounds
Table 10.5 (Continued)
Type (Subclass) Stem Structure
9. CNH type (bis-iodophenylalkanoates)
n = 0 to 5 carbon atoms alkylene chain, X = H, C,H,.
ecule for their high water solubility. To mini-
mize solution viscosity, all nonionic contrast
agents are designed to be small and globular
molecules. Substituent groups most fre-
quently found in nonionic radiopaque mole-
cules are, for example, hydroxyethyl, 2,3-
dihydroxypropyl, and 1,3-dihydroxypropyl.
These susbstituents are attached to the tri-
iodophenyl ring by the coupler groups, such
as amide (-40-NH-), reversed amide
(-NH-CO-), amino (-NH-), and so
forth. Depending on the nature of substituent
groups, hydroxyl groups from different sub-
stituent groups can have different hydrophi-
licities. For example, the hydroxyl groups in
metrizamide from a sugar molecule are highly
hydrophilic but unstable toward heat at
120°C. To avoid decomposition, metrizamide
is sterilized at low temperature and marketed
as lyophilized powder to be reconstituted with
sterile water just before use (401). On the
other hand, hydroxyl groups from hydroxyal-
kyl substituents are less hydrophilic and heat
stable at 120°C. Nonionic monomers and bis
compounds, such as iopamidol, iohexol, and
iotrolan, containing alkyl hydroxyl groups, are
heat stable and can be sterilized at 120°C for
2 h (401).
In designing improved nonionic contrast
agents, high water solubility is achieved by
masking and shielding the lipophilic regions of
Comments Fkf.
NH2
I
NH2
Synthesis and testing 383
the molecule (i.e., around the bulky iodine at-
oms and about both faces of the triiodophenyl
ring) with polar groups to achieve lateral and
facial hydrophilicity (204). High water solubil-
ity is necessary to minimize protein binding
and to achieve high biological tolerance. It is
difficult to predict the water solubility of a
molecule from the number of hydroxyl groups
it contains because hydrogen bonding and the
prevalence of multiple isomeric forms, for ex-
ample, can exert a strong influence on the mo-
lecular interaction with water. Nonionic con-
trast agents in mixed isomeric forms are more
water soluble than those present in only one
pure isomeric form, the only exception being the
pure L-ladoylform of iopamidol, which is more
soluble than the mixed isomeric forms (194).
Nonionic contrast agents are less anticoag-
ulant than ionic contrast agents (402-405).
Ranganathan et al. (313, 314) showed that
placing a heterocyclic ring substituent in the
5-position of the 2,4,6-triiodo-l,3-benzenedi-
carboxamide moiety can confer an unexpected
anticoagulant behavior to the nonionic con-
trast molecule.
6.3.2 Radiopacity. Radiopacity is depen-
dent on the number of iodine atoms in the
molecule, with more iodine atoms per mole-
cule yielding better images, provided that
other properties are equal (181). An ionic con-
532 Radiopaques

Table 10.6 Potential Radiopaques: Polymeric Substituted 2,4,6-TriiodobenzoicAcids

Compound Uses Comment Ref.
1. N = (A, raphy Synthesis
7

COOH

Roentgenography of
gastrointestinal
tract
7

where X = -N(Ac)- or -CO(NCH3)-;

3. Mixtures of iodobenzoic acid derivatives and cellulose

derivatives
r 7

$o-Jx
1
, ZY
Z = H, halo, C,-C,, alkyl, cycloalkyl, lower alkoxy, cyano; R = C,- A mixture of the 384
C,, alkyl, cycloalkyl or halo-lower alkyl, fluoro-lower alkyl, aryl, derivativeeused for
lower alkoxy, hydroxy, carboxy, lower-alkyl carbonyl or lower oral or retrograde
alkoxy-carbonyloxy; or (CRlR&-(CR&R4)m Q or (CR,R&- examination of GI tract
C=C-Q; R,, R,, R,, R4 = lower alkyl or halo-alkyl; X = 1 3 ; y =
1-4; n = 1-5; m = 1-15;p = 1-15;p = 1-10; and Q = H, lower
alkyl, alkenyl, alkynyl, lower alkylene, aryl or aryl-lower alkyl.
6 Organic Iodine Compounds 533

Table 10.7 Potential Radiopaques: Substituted 3,5-Dilodo-4(W-pyridones

Y Uses Comment Ref.

-CH,CO,R [R = Bu, CH,Ph, C,-Clo Synthesis 343,352,385
alkyl, CH,OAc, CH,OCOCMe,,
CH,CCI,]
-(CH,),CO,R [R = H, Me, Et, Bu, Urograph~, Synthesis (18 386
Am, octyl, CH,CH,NMe,, l~mphograph~ compounds
CH2CH,0H, NH,] prepared)
-CH,CONH(CH,),NR1R2 [n = 2,3; R1 Synthesis 387
1 2
= R2 = Me, Et, R R = (high toxicity)
CH,CH,OCH,CHz]
-CH2CH2R Syn thesis 387
(high toxicity)

trast molecule can dissociate in solution into
an iodine-containing anion and an iodine-free
cation, thus reducing the ratio of three iodine
atoms per molecule by a factor of 2 to a ratio of
1.5 iodine atoms on average per ion (177,180).
All the ionic monomers are therefore known
as iodine ratio 1.5 contrast agents. The mono-
acidic bis compound ioxaglate with one car-
boxylic acid group and six iodine atoms in the
molecule is a ratio 3.0 contrast agent. The
nonionic monomers, which contain no ioniz-
able groups and do not undergo dissociation in
solution, are ratio 3.0 contrast agents, and
their dimers or bis compounds ratio 6.0 .
agents. Higher ratio contrast agents with
higher iodine content per particle will give bet-

Table 10.8 Potential Radiopaques: Monoiodophenyl Derivatives

(1) Uses Comment Ref.

-CO,XO,CR [X = (CH,),, (CH,),, CH,CHMe, Myelography Synthesis 388
or (CH,),; R = Me, Et, Pr, i-Pr, Bu,
(CH,),Me, (CH,),Me, or CH,OMel
-CO,(CH,),O,CR [n = 2 4 , R = Me, Et, Pr, Myelography Synthesis 389
i-Pr, (CH,),Me, (CH,),Me, (CH,),Me,
CH,CHMe, or CH,OMe]
-X-OCO& [X = (CH,),, (CH,),, Myelography, Synthesis 390
CHMeCH,CH,, CH,CHEtCH,, or l~m~hogra~h~,
CH,CHBuCH,; R = Et, i-Pr, Bu, i-Bu, bronchography,
pentyl, hexyl, octyl, decyl, CHMeC,H13, salpingography
CH,CH(Et)Bu, CHMeCH,CH(CH,),,
CH(Et)Pr, or CH,CH,OMe]
534 Radiopaques

Table 10.9 Miscellaneous Potential Radiopaques

Compound Uses Comments Ref.
ICH,SO,NRR1 [R = H, Me; R1 = Myelography Synthesis 391,392
CH,CH(OH)CH,OH, CH(CH,OH),,
CH,CH,OH, CH,CH,OCH,CH,OHl
I
I
RCOC = CCOR
I
I Synthesis
[R = OH, O(CH,),OMe, O(CH,),OEt,
(OCH,CH,)OMe, (OCH2CH,)20Et,
O(CH,),Me, O(CH,),CHMe,, NH(CH,),OH,
NHCH,CH(OH)Me, NHC(CH,OH),Me,
N(CH,CH,OH),, NHCHCO~H,or
NMeCH,CO,Hl
BuNHCONHR (R = 2,3,5,6-tetraiodo-p- Radiography of Synthesis
tolylsulfonyl) pancreas and
prostate gland
OR
I

(R = glycosyl) Urography 185

Synthesis, toxicity 395
studies

Cholescystography Synthesis

Cholecystography Synthesis

I'
[R1 = NH,, N = CHNMe,; R = NH,, OH,
NMe,, NEt,, NBu,, morpholino, piperidino,
NHCH,CH,OH, N(CH,CH,OH),, or
NMeCH,(CHOH),CH,OHl
6 Organic Iodine Compounds
Table 10.9 (Continued)
Compound
~ O N R ~ R ~
Z 3
[NRR1, NR R = NHMe, NHCH2COzH,
NHCH(Pr)CO,H, morpholino, 2-carboxy-l-
pyrrolidinyl, NHCHEtCH2C02H,
NMeCH,C02H, NEtCH2C02Hl
+ +
Me2N(CH ) NMez. 2X-
I 7
R R'
[n = 2,4, 6, or 10; R, R1 = H, ethylacetyl, 3-
iodobenzyl, 2,4,5-triiodobenzyl, 5-amino-2,4-
didipdobenzyl, 3-amino-2,4,6-triiodobenzyl;
X = C1, I]
ter X-ray image and resolution. Radiopacity is
a physical property, affected not only by the
atomic number but also by the localization
and concentration of contrast medium in the
organ.
Gadolinium chelates provide contrast en-
hancement in CT and also in MRI. Gadolin-
ium has a higher atomic number than that of
iodine and can attenuate X-rays more effi-
ciently than iodine at equivalent mass concen-
tration, although iodinated contrast media
yield better resolution at higher concentra-
tions (91).
6.3.3 Acidity. The inductive effect of the
iodine atoms in the molecule makes the sub-
stituted 2,4,6-triiodobenzoicacids stronger ac-
ids than the substituted 2,4,6-triiodophenylor
triiodophenoxy alkanoic acids. The pKa values
of many ionic contrast agents were measured
by Felder et al. (263). The nonionic contrast
agent ioparnidol has a pKa value of 10.7 for the
nitrogen proton next to the triiodiophenyl
ring, but the molecule is practically undissoci-
ated at the physiologic pH 7.0-7.5 (194).
6.3.4 Electron Density. Substituent groups
attached to the triiodinated phenyl ring can
donate electrons to the ring or withdraw elec-
Uses Comments Ref.
Synthesis 398
Binding to cartilage Synthesis 399
trons from it. Substituent groups attached to
the ring through oxygen and nitrogen atoms
will donate electrons to the ring and those at-
tached through the carbonyl carbon atoms
will withdraw a-electrons from the ring. So-
vak (203) related the decrease in toxicity of a
series of model compounds to the decrease in
a-electrons in the ring. The following groups
are listed in the order according to their in-
creasing ability to withdraw a-electrons from
the ring: methyleneoxy (4-CH,-0-1 < methyl-
eneamino (+CH,-N-) < reversed amido (4-
NH-CO-) < alkyloxy (+O-CH,-) < carbamoyl
($40-NH-) group. Among the nonionic con-
trast media, listed in Table 10.3, iosimide (7g)
and iotriside (7h)are derivatives of trimesic
acid, which belong to the CCC subclass and
have the lowest a-electron density in the ring.
Gries and Miitzel(205) gave the number of
a-electrons in the benzene ring, in addition to
the six benzene electrons, for a series of model
compounds ranging from subclass NNN to
subclass CCC. These a-electron values are
given for each subclass in parentheses as fol-
lows:
"NNN" (a: 0.1088) > "CNN" (a: 0.1077)
> "CCN" (a:0.0947) > "CCC" (a: 0.0815).

This means that the model compound de-
rived from triiodotrimesic acid (subclass CCC)
contains fewer T-electrons than the model
compound derived from isophthalic acid (sub-
class CCN) and thus has a greater biosafety
and a higher intravenous median lethal dose
(LD,,) than that of the latter. Average values
of neural tolerance, expressed in mg iodine per
kg body weight, for these model compounds
are given in parentheses below:
> "CCN" (11.4 mgI/kg)
These values support the hypothesis that
associates the least toxicity of an iodinated
contrast molecule with the smallest +electron
density in the benzene ring.
6.3.5 Hydrophilicity and Solubility. Con-
trast agents for angiography are by necessity
administered intravascularly in large doses
and, for this, high water solubility is required.
Agents for oral cholecystography need an op-
timum oil-and-water solubility so that upon
ingestion, the molecule can be absorbed and
transported across the intestinal cell mem-
brane, from blood to the liver, and concen-
trated in the gall bladder. Agents for myelog-
raphy may be oil-soluble or water-soluble
compounds. The molecular requirements for
different contrast agents differ and do not nec-
essarily focus on the same substituent groups.
Solubility of contrast agents is determined
mainly by the presence of hydrophilic groups
(180, 209). In an ionic contrast molecule the
carboxyl group in the R group in (1)confers a
high water solubility to the molecule; and an
acylamino, alkylcarbamoyl, or hydroxylated
alkylamino group in the X or Y group in (1)
modifies such properties as its hydrophilicity,
toxicity, distribution, and excretion. Thus,
substituted triiodobenzoates and triiodo-
isophthalamates are highly water soluble
compounds. Solutions with concentrations as
high as 90%can be achieved with iothalamates
and metrizoates. These highly soluble con-
trast agents are also strong acids with pK, val-
ues less than 3. In nonionic contrast molecules
Radiopaques
the hydroxyl groups, hydrogen bonding, and
hydrophobic associations determine the water
solubility (171, 182, 2021, which may be fur-
ther enhanced by introducing more hydro-
philic groups (199).
Asymmetry in the substitution of contrast
molecules also influences the solubility (9).
The sodium salt of diatrizoic acid with a sym-
metrical 3,5-diacetamido substitution has rel-
atively low water solubility and concentra-
tions greater than 50% cannot be obtained.
The solubility is enhanced when one of the
acetamido groups is replaced with a N-meth-
ylcarbamoyl, N-methylacetamido, or acet-
amidomethyl group, as in iothalamate, me-
trizoate, or iodamide. N-Methyl substitution
can substantially increase the water solubility
of nonionic contrast agents (199), but the hy-
droxyl groups have to be evenly distributed to
mask the lateral and facial lipophilic regions
in the molecule, distinguished conceptually as
lateral hydrophilicity and facial hydrophilicity
(204).
Oral cholescystographic agents must pos-
sess an optimum oil-and-water solubility for
duodenal absorption. Substituents, such as
carboxyl, alkyl, or aralkyl groups, can impart
both hydrophilicity and lipophilicity to the
molecule. Iopanoate, ipodate, and tyropano-
ate, for example, are substituted triiodophenyl
alkanoic acids and will meet this requirement.
The chain length of the substituent can affect
the quality of the image. Epstein et al. (406)
observed that in a series of iodinated p-hy-
droxyphenylalkanoic acids, optimal visualiza-
tion of dog bladder was achieved with five to
eight carbon atoms in the alkanoic acid chain.
Felder et al. (291) reported that the insertion
of a methyl group between the oxygen and the
a-carbon in the series of substituted triiodo-
phenoxyalkoxyalkanoicacids can improve oral
absorption, biliary excretion, and gall bladder
visualization.
6.3.6 Chemotoxicity. The development of
modern contrast agents began with the obser-
vation by Wallingford et al. (219) that iodo-
benzoic acids have very low toxicity. Iodina-
tion, substitution, and acetylation can modify
the acute toxicity of substituted benzoic acids
(209,210). For example, amination of sodium
benzoate decreases toxicity, and the intrave-
6 Organic Iodine Compounds
nous median lethal dose (LD,,) values of
3-aminobenzoate and 3,5-diaminobenzoate
(i.e., 3270 and 2600 m a g , respectively) in
mice are higher than that of benzoate (1440
mg/kg). Acetylation decreases toxicity, as is
shown by the even higher LD,, values of 3-ac-
etamidobenzoate and 3,5-diacetamidobenzo-
ate (3400 and 5580 mgl kg) than of the corre-
sponding amines given above. In the series of
3-acylamino-2,4,6-triiodobenzoates,the de-
toxifying effect of substitution by acylation
reaches a maximum of two carbon atoms in
the acetyl group and further lengthening of
the acyl chain causes an increase in toxicity
(219).
Iodination may either decrease or increase
the toxicity, depending on whether the parent
compound is acetylated or unacetylated. Both
3-acetamido-2,4,6-triiodobenzoate and 3,5-di-
acetamido-2,4,6-triiodobenzoate(LD,,: 8300
and 14,000 mg/kg) have lower toxicities than
their corresponding noniodinated parent com-
pounds. A fully substituted benzene ring fur-
ther decreases the toxicity. 3,5-Diacetamido-
2,4,6-triiodobenzoate is the least toxic of
the series and is available commercially as
diatrizoate sodium and meglumine salts for
clinical use in angiography, pyelography,
urography, and other related roentgeno-
graphic procedures.
Although contrast media are remarkably
safe, when injected intravascularly in high
concentrations as a bolus, the blood is replaced
for a very brief period with the contrast me-
dium. Such high concentrations can produce a
myriad of dose-dependent pharmacological ef-
fects, often manifested as undesirable side ef-
fects. Rosati and de Haen (407) classified these
toxic effects as (1)chemotoxicity in distinction
to molecular toxicity, arising from unique
structural features that show affinity to bind-
ing with biomacromolecules; and (2) osmotic
toxicity, attributed to many side effects caused
by the considerably higher osmolality of ionic
contrast media relative to that of body fluids.
These authors correlated the LD,, values of
ionic and nonionic iodinated contrast media
for uroangiography directly with their osmo-
lality. Nonionic contrast agents, because of
their lower osmolality, high water solubility,
and a fully substituted benzene ring, do not
combine with proteins or macromolecules and
are considerably less toxic than ionic contrast
media. Contrast agents, regardless of their
ionic and osmolality differences, in the pres-
ence of X-ray irradation, may cause chromo-
some aberrations (408-411). Iodinated con-
trast agents in combination with X-ray
radiation can develop synergistic cytotoxicity,
possibly mediated by energetic photoelec-
trons, and this cytotoxicity increases with io-
dine concentration (410). Norman et al. (413)
showed that in iodine dose-enhancement ther-
apy for brain tumors, the iodine contrast me-
dia help localize the tumor and increase the
absorbed radiation dose.
The contrast bis compounds, in general,
have lower toxicity than that of the corre-
sponding monomers (258). The toxicity of
hexaiodinated ionic bis compounds increases
with increasing length of the alkylene bridge
and also with increasing length of the sub-
stituent group at position 5 of the triiodophe-
nyl ring (224,414). Bis compounds with open
positions in the triiodophenyl rings linked by a
polyoxymethylene bridge have a higher intra-
venous toxicity in mice than those with the
fully substituted benzene rings. Replacement
of the substituent n-butyramido group with a
butyrolactamyl group decreases the toxicity of
the bis compounds linked by a short alkylene
bridge but increases the toxicity of those
linked by a long alkylene bridge. Introduction
of one or more oxygen atoms into the alkylene
bridge greatly reduces the toxicity. For this
series of bis compounds, optimum tolerability
was achieved in the compound formed by join-
ing two molecules of 3-amino-5-acetylamino-
methyl-2,4,6-triiodo-benzoic acid with tetra-
oxahexadecane-dicarboxylic acid dichloride
(224). Ioxaglate is an asymmetric bis com-
pound, consisting of a dipeptide bridge linked
to two dissimilar monomers, one ionic and the
other nonionic (189). Ioxaglate is a ratio 3.0
contrast agent and has lower osmolality and
lower toxicity than that of the ratio 1.5 sym-
metric ionic bis compounds.
Hexaiodinated nonionic bis compounds are
ratio 6.0 contrast agents and owe their low
osmolality, high hydrophilicity, and high bio-
logical tolerance to 8-12 hydroxyl groups in
the molecule. Iotrolan containing 12 hydroxyl
groups has an osmolality isotonic to blood. In
concentrated solutions nonionic contrast me-
538
dia may undergo molecular association (i.e.,
hydrophobic interaction), to form relatively
small aggregates (quasi-dimers or quasi-
oligomers), thus giving a lower osmolality
than that in dilute solutions (202,415).
6.3.7 Protein Binding and Excretion. The
capacity of contrast agents to bind serum pro-
teins is related to certain structural features
(414, 416, 417). Contrast agents with a fully
substituted benzene ring show little or no pro-
tein binding and those having an open position
5 in the ring bind readily to serum protein.
Protein binding favors bilitropism (i.e., being
hepatotrophic, excreted through the liver)
rather than urotropism (i.e., being nephrotro-
phic, excreted through the kidney). The rela-
tive magnitude of biliary and urinary excre-
tion of the contrast agent, also known as the
B/U ratio, is determined by the structural
features of the molecule, the dose, and the
patient's condition. Fumagalli et al. (418,
419) observed that N-alkylation promotes
bilitropism.
According to Hansch (420, 421), protein
binding is nonspecific and occurs with many
compounds with sufficient lipophilicity. The
structural requirements for bilitropic agents
are sufficient hydrophobicity and an open po-
sition 5 in the triiodophenyl ring. Knoefel and
Huang (422) observed that protein binding
correlates with increasing toxicity in a series
of substituted iodinated benzoic acids. In
hexaiodinated ionic bis compounds, an in-
crease both in the length of substituents and
in the length of the alkylene bridge enhances
hydrophobicity and increases the protein
binding and the B/U ratio (423). Higher B/U
excretion ratios are observed in bis com-
pounds with open position 5 in the phenyl ring
than in those that are fully substituted. Intro-
duction of one or more oxygen atoms into the
alkylene bridge increases water solubility and
reduces toxicity (189).
Nonionic contrast media, by virture of the
large number of hydroxyl groups, 4 to 6 in
monomers and 8 to 12 in bis compounds, are
highly water soluble. These hydroxyl groups
are evenly distributed throughout the con-
trast molecule and leave no lipophilic regions
in the molecule free to bind with proteins. The
hexaiodinated nonionic bis compound iodixa-
Radiopaques
nol, similar to others of this class, is not me-
tabolized and is excreted in the urine un-
changed by the kidney (270,258).
6.3.8 Osmolality and Viscosity. Ionic con-
trast media exert osmotic pressure in solution,
and high concentrations of ions cause adverse
reactions in the patient (181, 424). The os-
motic pressure of a solute is measured in
terms of osmolality, in relation to the pressure
exerted by a gram-molecular weight of an
ideal un-ionized substance dissolved in 1 kg of
water (425), measured as mOsm or Osmkg
H,O. Ionic and nonionic contrast media are
referred to as high osmolality contrast media
(HOCM) and low osmolality contrast media
(LOCM), with values of 1600 and 600 mOsm/
kg, respectively; for reference, the osmolality
of the body fluids is about 300 mOsm/kg H,O.
Ioxaglate, the monoacidic asymmetric dimer,
belongs to the LOCM category. The hexaiodin-
ated nonionic dimers, such as iodecol, iotasul,
and iotrolan, may have an even lower osmolal-
ity than that of the nonionic monomers (203,
258, 415 mOsmkg). Krause et al. (415) mea-
sured the osmolality of a number of commer-
cial products of X-ray contrast media and
ranked their osmolalities at 300 mg I/mL in
increasing order as follows: iotrolan << ioxa-
glate < iopromide < iopamidol < ioversol =
iohexol < iopentol << diatrizoate. The physi-
cochemical parameters of the commercial
products of iotrolan (iotrolan-280, iotrolan-
300, iotrolan-320) are, respectively: iodine
concentrations 278,296,321 (mg I/mL); iotro-
lan concentrations 0.595,0.632,0.687 (g/mL);
osmolalities 272,291,317 (mOsm/kg);product
densities at 20"C, 1.333, 1.353, 1.379 (g/mL);
and product viscositiesat 20"C, 13.4,17.4,25.3
(mPas). At 37°C the density is decreased only
slightly but the viscosity is reduced by more
than 50%. At 37°C iotrolan-300 has a viscosity
of 8.1 m P a s (426). In comparison, the blood
has a viscosity of 4 mPa.s. To include ionicity
with osmolality, Hardeman (427) suggested a
more exact nomenclature of ILO (ionic low-
osmolar), NIL0 (nonionic low-osomolar, and
IHO (ionic high-osmolar) in referring to con-
trast media.
Osmolality and viscosity depend on the
ionic character and structure of the contrast
molecule (10, 11, 181, 428). Alm6n (181)con-
6 Organic Iodine Compounds
cluded from physicochemical principles that
both osmolality and viscosity of a contrast me- versol, iopamidol, and iohexol, can be analyzed
dium can be reduced if nonionic and spherical
polymeric molecules are involved. Polymers
can have more iodine atoms per molecule than
can monomers and a lower osmolality. A
prominent example of more iodine atoms and
low osmolality in a molecule is the monoacidic
asymmetric dimer ioxaglate (203). Spherical
molecules show less resistance to flow than do
linear molecules and tend to have lower vis-
cosity.
- Nonionic molecules make no contribu-
tion to electrolyte concentration and show no
osmotic toxicity. The LD,, of ionic diatrizoate for analysis after dilution (434). HPLC is a
when injected into the subarachnoid space is
about 50 mg I/kg in mice; in comparison, the
LD,, values for nonionic contrast agents, me-
trizamide and iohexol, are in excess of 1500mg
I/kg (182). The remarkable safety of the early
nonionic contrast agents provides a stimulus
for the search for improved contrast agents,
especially as dimers, trimers, polymers, and
nonionic compounds. Nonionic iodinated
organic compounds, such as triglycosyldi-
iodobenzene, 2,4,6-triiodo-3-acetamido-5-N- originally designed for the analysis of iopam-
methylcarboxamido-phenyl-D-glycopyrano-
side (186) and others, have been synthesized
for clinical tests a s potential contrast
agents.
Iodinated nonionic dimers iodixanol, iode-
col, iofratol, and iotrol (or iotrolan) have re- pamidol in plasma and urine samples are in-
spectively lower osmolalities (200, 320, 140,
and 320 mOsm/kg H,O) and higher viscosities
(8.7, 7.2, 8.5, and 8.1 m P a s at 37°C) than
those of iodinated nonionic monomers. Felder
and de Haen (429) found unexpectedly that
solution mixtures of iodinated nonionic mono-
mers and dimers, such as iomeprol and iofra-
tol, have a lower toxicity and a lower viscosity HPLC to measure and predict the lipophilici-
than those of either of the components and can
supply a favorable iodine delivery rate
through less invasive catheters during the in-
jection, but have an intermediate osmolality
compared to that of the pure solutions of the
monomer and dimer components.
6.4 Analysis
A contrast medium may be analyzed by con-
ventional means for its iodine content and
functionality or by capillary electrophoresis
(CE) and high performance liquid chromatog-
raphy (HPLC) for its components. Nonionic
contrast media, such as iosimide, iopentol, io-
in a borate buffer by capillary electrophoresis
(430, 431). Borate ions combine with the 1,2-
diol and l,3-diol groups in the molecule, to
form negatively charged complexes that can
migrate in an electric field. Iohexol in serum
can be measured and quantitated by CE
against an internal standard after deprotein-
ization with acetonitrile (431, 432) or by
HPLC after deproteinization with tetrahydro-
furan (433). A serum sample of iohexol may
also be injected directly onto an HPLC column
versatile analytical technique and can qualita-
tively and quantitatively assay contrast
agents as intact molecules, geometric isomers,
and metabolites. For example, HPLC can sep-
arate isomeric iohexol into endo and exo forms
(1:3) (267). An automated HPLC system, with
online dialysis and column-switching to enrich
dialysate, can analyze samples of iopentol in
human serum or whole blood (433). An auto-
mated analytical HPLC, using a workstation
idol, can be adapted for the analysis of other
contrast agents (435). The early HPLC meth-
ods relying on elaborate sample purification
and higher column temperature for analysis
and separation of ioversol, iomeprol, and io-
convenient for routine assay (436, 437). An
improved method using reversed-phase HPLC
and a new mobile-phase system can routinely
analyze samples of diatrizoate, iopamidol, and
iohexol in the serum and testes of the mouse to
study the uptake and clearance kinetics (432).
Alonoso-Silva et al. (438) used reversed-phase
ties or partition coefficients of nonionic con-
trast agents. Liquid-liquid partition coeffi-
cients measured by HPLC correlate well with
the values calculated by the Hansch method
(439).
Contrast agents in plasma may also be de-
termined by spectrophotometric procedures
(440, 443) or by radiotracer techniques using
or l3lI-labeled materials (442-446).
Felder et al. (447) measured the concentration
of water-soluble radiopaques by colorimetric
determination of free aromatic amine function
using the Bratten-Marshall reaction. Hart-

mann and Roepke (448) reported methods for
determining the purity and stability of iodine-
containing contrast agents of the aminoben-
zoic acid series. In vitro analysis of iodine con-
tent of contrast media can be carried out by
the Sandell-Kolthoff reaction (189) or by in-
ductively coupled plasma atomic emission
spectrometry. The iodine concentration in tis-
sue samples can be determined by the fluores-
cent excitation method by the K, and Kpchar-
acteristic X-rays generated from irradiation
with a 241Am source (449, 450). This method
can also be applied to in viuo measurement of
hepatic iodine concentration after administra-
tion of a radiopaque (451).The range of iodine
determination is 0.05 to 40 mg/mL, with an
accuracy of approximately 210%. Measure-
ments based on function groups or iodine at-
oms represent the concentration of these
groups directly and not necessarily the con-
centration of the intact contrast agent.
6.5 Pharmacology
Contrast media by necessity are relatively
nontoxic. Adverse reactions accompanying
their use vary and usually decrease in inten-
sity and complexity in the order intracerebral
> intravascular > oral route of administra-
tion > topical application (442-457).Contrast
molecules that bind proteins, biomacromol-
ecules, and enzymes are more toxic than those
that do not. The toxicity decreases with in-
creasing hydrophilicity for different classes of
contrast media: oral cholecystographic media
> intravenous cholangiographic media >
ionic triiodinated uroangiographic media >
monoionic hexaiodinated molecules > non-
ionic triiodinated contrast media > nonionic
hexaiodinated contrast media. Median lethal
dose (LD,,) is an indication of systemic toxic-
ity, even though it lacks predictive value (458).
The intravenous LD,, values of the last four
classes of contrast media are in the range of
tens of grams per kilogram body weight when
tested in rats or mice. The opacifying atom
iodine has an intravenous LD,, in animals of
0.8-1.0 g/kg; the intravenous LD,, values (ex-
pressed in g Iikg in mice) of uroangiographic
contrast agents increase, for example, from 7
for ionic iothalamate to 13.8 for monoacidic
dimer ioxaglate and then to even greater val-
ues for nonionic contrast agents, such as 18.1
Radiopaques
for metrizamide, 22.1 for iopamidol, and 24.3
for iohexol(178). The subarachnoid LD,, val-
ues of ionic diatrizoate and nonionic metriz-
amide and iohexol differ by a factor of 30,
whereas their osmolalities differ by a factor of
only 2 (459). The oral cholecystographic
agents have considerably higher toxicity but
the side reactions are generally restricted to
gastrointestinal symptoms.
Procedures in angiocardiography, intrave-
nous digital subtraction angiography, and
rapid-scan CT require rapid and multiple in-
travascular injections of low osmolality con-
trast media, causing significant hemodynamic
changes (460,461). A bolus injection can be as
short as 2 s (460,461),and a volume of up to 4
mL I/kg body weight may be injected (462).
According to Golman and Alm6n (463), the
toxicity of contrast media is dependent on in-
jection rate and dose, and increases with ei-
ther an increase in the dose or an increase in
the rate of injection, or both. The dose-re-
sponse curve is typically an S-shaped curve
with dose as abscissa and percentage mortal-
ity as ordinate, and this curve shifts in its en-
tirety along the dose axis according to the rate
of injection. The curve begins at a lower dose
level with rapid injection, indicating greater
toxicity, and shifts to a higher dose level with
slow injection, indicating greater safety. Al-
though delivery by slow injection is safer than
by rapid injection, rapid injection of contrast
medium gives a better image quality. de Haen
et al. (458) suggested that the mortality ratio,
which incorporates both volume factor and in-
jection time factor in its definition, can serve
as a better predictor than the median lethal
dose (LD,,) for acute intravenous toxicity of
contrast agents. The mortality ratio is a func-
tion of three parameters: the osmotic load (re-
ferring to excess osmolality associated with
isotonic volume), the volume of contrast me-
dium, and the duration of injection. The end-
point is death, measured by the time between
administration of the dose and death of the
mouse, and is closely dependent on the rate of
injection of the contrast medium. This sug-
gests that the mechanism of contrast medium
toxicity may involve early damage and late
consequences (458).
The pharmacological aspects and toxic re-
actions of contrast agents have been compre-
6 Organic iodine Compounds
hensively reviewed by Hoppe (464), A l m h
(457), Ansell (465), Berk and Loeb (466), and
Speck et al. (258).
6.5.1 The Cations. Iodinated ionic contrast
agents (ratio 1.5) used in angiography, urog-
raphy, and intravenous cholangiography are
substituted 2,4,6-triiodobenzoic acids. These
agents are formulated for intravenous admin-
istration as solutions of salts, ionized at phys-
iological pH. Their sodium salts have high
water solubility, and solutions of 5O-gO% con-
centration, corresponding to an iodine content
of about 300 to more than 400 mg I/mL, can be
prepared for angiographic use (10, 11). Ionic
contrast media with sodium concentrations of
118-370 mEq/100 mL rarely produce ventric-
ular fibrillation in perfused rabbit heart (467)
but sodium ions in excess amount are cardio-
toxic (468). The sodium ion concentration of
ionic contrast media is many times higher
than that of plasma. Their toxicity in high con-
centrations is attributable not only to high 0s-
molality but also to the effects of their cation
and anion components (9,10,34,36-43). The
cations in regular use in contrast media are
N-methylglucamine, sodium, calcium, and
magnesium.
N-Methylglucamine (meglumine) ion has
the following structure:
H OHH OH OH
I I I I
I l l 1
H H OHH H
Meglumine salts of contrast media such as
diatrizoic, iothalamic, and metrizoic acid have
higher viscosities than those of the corre-
sponding sodium salts but are better tolerated
and less toxic. The high viscosity of the meglu-
mine salts can be reduced by mixing with the
sodium salt of the corresponding acid (469).
Mixed salts of diatrizoate are marketed under
various proprietary names (223). During cor-
onary angiography, sodium ions in contrast
media reduce the risk of ventricular fibrilla-
tion and reverse the reduction of contractile
force of the myocardium. A recent study
showed that meglumine diatriazoate without
Nat caused a higher frequency of ventricular
fibrillation in isolated rabbit heart than did
diatrizoate with 77 or 154 mmol of Na+ added
(470). Also, the meglumine salts of ionic con-
trast media inhibit in uitro leukocyte phagocy-
tosis more than do the sodium salts (471).
Other organic bases, such as diethylamine,
monoethanolamine, diethanolamine, trietha-
nolamine, and morpholine have low toxicity
and can be used in place of cations (9, 225).
The monoethanolamine salt of ioxitalamic
acid shows a toxicity intermediate between
those of sodium and meglumine salts (9). Di-
ethanolamine and morpholine form 1:l salts
with 3,5-diiodo-4-0x0-l(4H)pyridine acetic
acid, named iodopyracet (Diodrast) and Jodu-
ron (2251, respectively. Other cations such as
basic amino acids also form salts with the acid
form of contrast agents to reduce toxicity (472).
For example, ionic diatrizoate, iothalamate,and
metrizoate form salts with Tris, which is
2-amino-2-(hydroxymethy1)-1,3-propanediol, to
yield solutions of lower viscosity than that of the
corresponding meglumine salts. Tris is an ali-
phatic amine with a negative temperature-acid-
ity coefficient; that is, its pK, value decreases
about 0.025 unit with each degree Centigrade
increase in temperature. This property allows
the nonionic contrast agent iohexol to be formu-
lated in Tris buffer at physiologic pH (7.0-7.5) at
room temperature and to be heat sterilized at ,
120"C, when the pH falls below 5.5 to avoid de-
terioration. The product after sterilization will
return to its original physiologic pH at room
temperature for use in angiography without
causing discomfort in the patient; this would not
have been the case had the solution remained at
pH below 5.5 (178,473).Another approach with-
out using an arnine buffer is to adjust the pH of
iohexol in a citrate-EDTA buffer to less than 5
with carbon dioxide before sterilization (474).
In normal blood, sodium, potassium, cal-
cium, and magnesium ions are present in con-
centrations of 330, 20, 5-6, and 2-2.5 mg1100
mL plasma, respectively (475). The physiolog-
ical functions of the cations are both antago-
nistic and complementary to one another. So-
dium ions tend to increase the permeability of
cell membrane, whereas calcium ions counter-
act this effect. Calcium and magnesium ions
are physiological antagonists; magnesium
ions can exert a deleterious effect on the ner-
vous system if unaccompanied by the revers-
542
ible antagonistic effects of calcium ions. The
physiological interaction between these ions
suggests that the toxicity of sodium ions in
contrast medium can be modified by " the addi-
tion of small concentrations of calcium and
magnesium, although incorporation of potas-
sium ions affords no beneficial effect. To min-
imize the toxicity, an optimum concentration
of calcium and magnesium ions in the contrast
medium is essential and should be 2.5 times
their concentration ratio in plasma (i.e., the
ratio of concentration of calcium or magne-
sium ions to that of sodium ions). Incorpora-
tion of optimal amounts of calcium and mag-
nesium ions in solutions of sodium metrizoate
can increase the LD,, values by 80-100% in
the rabbit. Calcium ions alone lower the toxic-
ity of methylglucamine metrizoate in LD,,
tests in mice but not in the rabbit, whereas
magnesium ions alone cause an increase in
toxicity (475). A small amount of calcium ions
in methylglucamine metrizoate causes less
blood-brain barrier damage than when pure
methylglucamine metrizoate of identical io-
dine content is given alone (476). Also, addi-
tion of calcium salt inhibits metrizoate- and
ioxaglate-induced serum complement activa-
tion (477). Formulations of diatrizoate that
minimize calcium binding are advocated for
cardiac angiography (478).
Nonionic contrast media are intrinsically
ion-free but iohexol, iopentol, and iodixanol,
for example, contain cations at subplasma
concentration levels to avoid negative effects
on myocardium contractility, electric conduc-
tion, and red blood cell aggregation during cor-
onary arteriography. An oxygenated iohexol
solution (350 mg IImL) containing (in mM/L)
Na+ 30, Ca2+ 0.15, Kt 0.9, and Mg2+ 0.1,
when injected into the left coronary artery of a
pig, produces fewer electrocardiographic
and/or hemodynamic changes than pure io-
hex01or iopamidol(479-481).Compared to no
sodium or excess sodium, the addition of a
small amount of sodium to iohexol and iopen-
to1 lowers the risk of ventricular fibrillation
and causes the least decrease in the contractile
force of the isolated rabbit heart. A physiolog-
ical amount of sodium ions is important in test
solutions of 20% mannitol, ioxaglate, iopam-
idol, iotrolan, and sodium iopamidol to pre-
vent fatal arrhythmia (482). In cardiac angiog-
Radiopaques
raphy, optimal concentrations of Ca2+ and
M8+ additives are necessary to maintain
myocardium contractile function and reduce
adverse reactions to contrast media (483,484).
Thus, a balanced electrolyte formulation for
even low osmolality contrast media is essen-
tial to minimize ion toxicity (459,470,484).
6.5.2 Hyperosmolality. Ionic contrast me-
dia for angiographic use are hyperosmotic rel-
ative to plasma. The osmolalities of the substi-
tuted triiodobenzoate anions diatrizoate,
iothalamate, and metrizoate are approxi-
mately the same on the basis of equal iodine
content (485). Injection of hyperosmolar con-
trast media causes hemodynamic responses
that vary according to the rate and site of in-
jection (469). Slow injection into a peripheral
vein allows visualization of the collecting sys-
tems of an organ, and rapid injection of a more
concentrated solution allows visualization of
the vasculature of an organ or a region of the
body (469). Slow injection into the peripheral
veins as in urography and intravenous cholan-
giography produces hypotension because of a
lowering of peripheral vascular resistance
(486). Acute intravenous toxicity of contrast
medium is increased with the rate of injection
(184, 487-489). A large volume of hypertonic
contrast medium, when rapidly injected into
veins, arteries, and ventricles in selective &I
-
giography causes an elevation of the plasma
osmolality and a shift of water out of red blood
cells and out from extravascular spaces to the
blood, thereby increasing the circulating blood
volume and peripheral blood flow and affect-
ing various aspects of circulation (10,428,466,
468, 469, 490-496). The more concentrated
the injectate, the more intense the vascular
reactions, and the more precipitous the onset
(469,491).
Systemic hypotension, changes in the ac-
tivity of the smooth muscle cells in vessel
walls, aggregation and crenation of red blood
cells, and hypervolemia are examples of vascu-
lar reactions induced by the administration of
ionic contrast media. Agglutination and cre-
nation of red blood cells cause blood sludging
and reduce the cells' ability to undergo de-
formation, thus blocking the blood flow to
capillaries and leading to pulmonary hyper-
tension and related physiological responses
6 Organic Iodine Compounds
(495-498).The hypervolemia resulting from a
shift of water elevates the ventricular filling
pressure and increases the cardiac output
from increased ventricular stroke work. The
potassium ions released from shrinking and
crenating red blood cells contribute to the low-
ering of systemic vascular resistance and
blood pressure (499).
That osmolality is a major determinant of
hemodynamic effects is clear from the experi-
ments conducted by Kloster et al. (500), who
injected equiosmolar solutions of mannitol,
sodium, and meglumine salts of diatrizoate
and iothalamate, with an osmolality corre-
sponding to 4.5 to almost 8 times the osmolal-
ity of blood serum, directly into the left ventri-
cle of a dog and observed approximately
similar hemodynamic responses. D-Mannitol
is often used in osmotic effect studies on cells,
organs, and organisms as a reference (458) be-
cause it is inert, distributed in extracellular
space, and acts as individual molecules, not as
dilution-dependent aggregates. Hilal(493) re-
ported that the bradycardia, hypertension,
and accompanying vascular reactions attrib-
uted to intracarotid injection of contrast me-
dia could be reproduced by injection of hyper-
tonic solutions of sodium chloride of similar
osmolarities. By comparing the hemodynamic
effects from intracarotid injection of 50% so-
dium diatrizoate, 80% sodium iothalamate,
and equiosmolar solutions of sodium chloride
and dextrose, Cornell (501) concluded that the
sodium ions may play a greater role than hy-
pertonicity in causing these effects.
Toxic effects of sodium ions and hyperos-
molality associated with monomeric ionic con-
trast agents are lessened when dimeric and
trimeric contrast agents containing, respec-
tively, six and nine iodine atoms per molecule
are used. Both bis(iothalarnic acid) (i.e., iocar-
mic acid) and tris(iotha1amic acid) produce
fewer peripheral vasodilatation and reflex re-
sponses than does the monomer iothalamic
acid (9).
Ionic contrast monomers at a solution con-
centration of 300 mg I/mL have an osmolality
in the range of 1.5-1.7 Osmkg H,O. At the
same solution concentration, nonionic mono-
mers, monoacidic ionic dimer (ioxaglate), and
nonionic dimers have osmolalities of 0.6-0.7,
0.56, and about 0.3 Osmkg, respectively. If
made isotonic to body fluids, the isotonic ionic
monomer solutions have an iodine content of
70 mg I/mL; the nonionic monomer solutions,
150; the monoacidic ionic dimer ioxaglate so-
lution, 150; and the nonionic dimer solutions,
300. The osmolalities of nonionic dimers or bis
compounds are isotonic to body fluids and are
the lowest of all contrast media. Hyperosmo-
lality causes vascular pain. Sovak et al. (502)
estimated the concentration threshold for vas-
cular pain to be about 750 to 800 mOsm. Thus
if the osmolality of a contrast medium at a
concentration of 350 mg I/mL does not exceed
the pain threshold, the solution would not be
expected to induce pain in arterial arteriogra-
phy. Metrizamide at 550 mOsm/kg is essen-
tially painless in peripheral angiography (503,
504), and all the nonionic contrast media are
expected to behave similarly. Metrizamide has
an extremely low cellular toxicity and can be
injected at an iodine concentration of 380 mg
I/mL into the common carotid artery of guinea
pig without damage to the blood-brain barrier.
6.5.3 Adverse Reactions and Toxicity. The
toxicity of a contrast medium is attributed to
its molecular structure, solution property, for-
mulation, and the amount used as a dose. Tox-
icity may be further classified as chemotoxic-
ity, osmotoxicity, and ion toxicity (407, 459).
'
Chemotoxicity is related to a molecular struc-
ture that allows binding to proteins, leading to
interaction with biomacromolecules such as
enzymes, cell membranes, and cell compo-
nents. Osmotoxicity is attributed to the hyper-
osmolality of contrast media, and a marked
difference exists between the osmolalities of
ionic and nonionic contrast media. Ion toxicity
refers to adverse reactions as a consequence of
too high or too low ion concentrations in con-
trast media that interfere with cellular func-
tion. Contrast media formulations containing
citrate and EDTA buffers can interfere with
serum Ca2+and affect myocardial contractil-
ity function. Contrast media are transported
by the cardiovascular system and are excreted
by the kidneys. Any reactions to contrast me-
dia are known as adverse reactions.
Contrast media are injected intravascu-
larly into the patients in large volumes and
high concentrations. The size of the injectate,
together with its high osmolality and viscosity,
544
can produce an intolerant environment to
cells, producing abnormal physiological ef-
fects. Patient safety and efficacy of iodinated
contrast media are of great concern to health
professionals (505, 506). Many aspects of ad-
verse reactions to contrast media, such as eti-
ology (459,507,508),adverse reactions in the
patients (505, 506, 509-5181, and premedica-
tion to decrease adverse effects (519-522)
have been researched and reviewed.
In humans adverse reactions to contrast
media vary greatly in type and severity from
slight nausea or mild "hot flush" through a
broad spectrum of increasingly severe cutane-
ous, respiratory, neurological, and cardiovas-
cular disturbances that may, in extreme cases,
result in the sudden death of the patient (454-
457,459,465,469, 507). Ansari and Baldwin
(523) reported acute renal failure after urog-
raphy with meglumine diatrizoate, angiogra-
phy with meglumine and sodium diatrizoate,
oral cholecystography with iopanoic acid, and
cholangiography with iodipamide. Roylance et
al. (524, 525) reported the incidence of reac-
tions to urographic agents.
To review the incidence and severity of ad-
verse reactions to contrast media, a committee
on contrast media was formed under the aus-
pices of the International Society of Radiology
in 1969 (526). Based on the analysis of a total
of 112,003 cases of adverse reactions toward
contrast media from Australia, Belgium, Can-
ada, Norway, and the United States, Shehadi
(452) reported the nonfatal incidence of reac-
tions to be 2.3% for vascular studies, 5.65% for
intravenous urography, and 10.11% for in-
travenous cholangiography. The incidence
of reactions for total intravenous and intra-
arterial examinations was 4.95%, and the in-
cidence of fatal reactions was about 1110,000,
which is higher than the 1160,000 reported
earlier on the basis of retrospective studies.
The contrast agents used in the cases studied
were meglumine diatrizoate, meglumine
iothalamate, sodium diatrizoate, and sodium
metrizoate. These studies did not include non-
ionic contrast media and predated their use.
In intravenous cholangiography the fre-
quency of toxic symptoms was 12.6% for sin-
gle-dose medication and 8.16% for drip infu-
sion (452). In intravenous urography the
incidence of reactions was 5.38% for single bo-
Radiopaques
lus injection and 7.06% for drip infusion that
required a higher dose of contrast medium
(452). The incidence was higher with slow in-
jection over a period of 3 to 10 min than with
rapid injection performed in less than 2 min.
This suggests a reversal of the hitherto wide-
spread practice of administration of contrast
media. Although the cause of this low inci-
dence of reactions with rapid injection is not
explained, it is not unreasonable to assume
that less histamine is released (489).
In hypersensitive or allergic patients, idio-
syncratic reactions may occur from a dose of
contrast medium, and the overall incidence of
adverse reactions in patients with allergy is
about twice that in the general population
(452-455,527). Pretesting the patient by intra-
dermal, subcutaneous, or intravenous injection
of small doses has no value in predicting adverse
reactions (452-455, 525, 528) but testing by in
vitro leukocyte challenge has been reported to be
of some predictive value (529). Premeditation
with antihistamines, anticholinergics, and diaz-
epam (468) is less effective than that with corti-
costeroids (454,525).
Shehadi and Toni010 (453)in 1980 reported
the result of a survey on the safety of intravas-
cular ionic contrast media in more than
300,000 cases collected from the United
States, Canada, Europe, and Australia and
showed that the overall incidence of reactions.
was slightly below 5%, with neither significant
geographic or racial differences nor differ-
ences related to age, sex, and weight. Most of
the patients were in their third and fourth de-
cade of life.
Beginning in 1980s the use of low osmolal-
ity nonionic contrast media has significantly
reduced the severity and frequency of adverse
reactions to contrast media. The high cost of
nonionic contrast media becomes an economic
barrier to a complete replacement of the ionic
contrast media. The price ratio of nonionic to
ionic contrast media in 1989 was about 3.5-
6.5 times to 1 in Europe and about 13.2-23.6
times to 1 in the United States (531).Prescrib-
ing an ionic contrast medium to a patient in
the face of a better choice, on the basis of cost
consideration, can have serious ethical and
medical implications (532). Palmer published
an interim guideline that identified high risk
groups who should receive nonionic contrast
6 Organic iodine Compounds
media, that is, patients with previous reac-
tions to contrast media, patients with asthma,
allergy (not to drugs), renal and cardiac im-
pairment, diabetes mellitus, myelomatosis,
and sickle-cell anemia; poorly hydrated pa-
tients; and infants and small children.
The Royal Australasian College of Radiolo-
gists (RACR)survey included 109,546 cases of
mild. moderate, and severe reactions to intra-
venous ionic and nonionic contrast media
(533, 534). The report defined mild reactions
as all skin reactions requiring no therapy and
excluding sensation of heat; moderate reac-
tions as those requiring therapy but not con-
sidered at risk and no hospitalization, and se-
vere reactions as those requiring urgent
therapy and considered at risk and requiring
hospitalization. The incidence of mild, moder-
ate, and severe reactions in the high risk group
were 7.20, 2.70, and 0.36%, respectively, for
ionic contrast media, and 1.10, 0.10, and
0.03%, respectively, for nonionic contrast me-
dia. Incidences of moderate and severe reac-
tions in the high risk groups receiving ionic
and nonionic contrast media were 1 in 32 in-
jections and 1 in 718 injections, respectively.
This clearly demonstrates a significant advan-
tage for the nonionic contrast media.
The Japanese Committee on the Safety of
Contrast Media conducted similar studies
from 1986 to 1988 (535-537). The large scale,
nationwide clinical study included 337,647
cases. One-half of the group (50.1%) received
ionic contrast media and the other half
(49.9%) nonionic contrast media. The overall
prevalence of adverse reactions was 12.66% in
the ionic contrast media group, and 3.3% in
the nonionic contrast media group. The prev-
alence of severe adverse reactions was 3.13
and 0.22% in the ionic and nonionic contrast
media groups, respectively. The report de-
tailed the prevalence of adverse reactions re-
garding time of onset, patient sex and age, pa-
tient history on prior reactions to contrast
agents, allergy, underlying disease, premedi-
cation, injection mode, and dose. The preva-
lence of adverse reactions was the lowest in
the subgroup of patients receiving contrast
media by bolus and intravenous injection as
compared to by drip infusion and by bolus plus
drip infusion. The patient subgroup receiving
a dose ranging from 81 to 100 mL of contrast
media, ionic or nonionic, showed the lowest
prevalence of adverse reactions (537). The
study clearly demonstrates that nonionic con-
trast media can significantly reduce the prev-
alence of adverse reactions to contrast media.
Reducing the mild adverse reactions such as
nausea, vomiting, urticaria, itching, and heat
sensation can make contrast media examina-
tions more acceptable to the patient. Bett-
mann (537) noted that the report showed no
difference between the ionic and nonionic con-
trast agents with regard to mortality. Fareed
et al. (538) pointed out that both the Japanese
and the Australian survey reports on the
safety of contrast media did not include any
information on thrombotic complications.
The use of premedication with steroids and
HI and H, histamine receptor blockers can
significantly reduce the adverse reactions to
high osmolality contrast media. There are sug-
gestions to extend their use to modify even the
reactions to low osmolality contrast media.
Pretesting of contrast media reactors by in-
jecting 1 mL of the nonionic contrast medium
has also been reported (539).
Greater use of nonionic contrast media for
routine X-ray procedures is clearly the trend
of the future. It was estimated that in 1991,
approximately 69% of the procedures per-
formed in the United States used nonionic
'
contrast media compared to about 66% in Eu-
rope and more than 80% in Japan. In 1994 the
percentage increased to about 80% in the
United States, 75% in Europe, and 92% in Ja-
pan (540). In the United States, in 1994 as
many as 18 million patients received intravas-
cular contrast media, and an estimated 170
million contrast medium-enhanced radiologic
studies were performed from 1978 to 1994
(541).
Spring et al. (541) analyzed the reports of
adverse drug events (ADE) of iodinated con-
trast media, filed with the Spontaneous Re-
porting System and MedWatch Program of
the U.S. Food and Drug Administration (FDA)
from 1978 through 1994. In this 17-year pe-
riod, there were 22,785 reports (86.5%)of mild
or moderate ADEs, 2639 reports (10.0%) of
serious nonfatal ADEs, and 920 reports (3.5%)
of deaths describing 850 fatal ADEs. Report-
ing to the FDA Spontaneous Reporting Sys-
tem and MedWatch Program is voluntary. Ad-

verse drug event (ADE) is the term used to
avoid contributing causation and to encourage
reporting of adverse consequences from pro-
fessional practice. When these events are spe-
cifically attributed to the drug, they are called
adverse drug reactions (ADR).To identify any
changes in the frequency of reporting between
ionic and nonionic contrast media, the authors
arbitrarily divided the 17-year period into an
earlier period (1978-1986) before FDA ap-
proval and wide use of nonionic low osmolality
-
contrast media (LOCM) and a later period
(1987-1994), when the use of nonionic LOCM
rose steadily over time after approval. In 1994
the use of nonionic LOCM exceeded ionic
HOCM by 2:l. For ionic and nonionic contrast
media the most common ADEs (urticaria, dys-
pnea, vomiting, facial edema, and hypoten-
sion) ranked similarly, and the intravenous
and intraarterial routes (97.4%)were the pre-
dominant routes of administration. The seri-
ous nonfatal ADEs were most often related to
serious cardiorespiratory symptoms. Myelo-
graphic ADRs (e.g., seizures, headaches) from
intrathecal administration of contrast media
differ from the most common ADRs men-
tioned above and were treated separately. The
authors noted the prevalence of severe nonfa-
tal ADEs to be 0.044% for nonionic LOCM and
0.26% for ionic HOCM, comparable with the
published values (537, 538) and also noted
that the number of ADEs reported did not sig-
nificantly decrease since the introduction of
nonionic LOCM.
Mortality as a direct consequence of use of
iodinated contrast media is a rare occurrence.
Hatayama et al. (535) observed two deaths
among 337,647 cases in which iodinated con-
trast media were injected intravenously.
Spring et al. (542) showed that 37% more
deaths were reported each year in 1987-1994
than in 1978-1986. Most of the increase was
associated with the use of nonionic contrast
media. In 1978-1987, 376 deaths were re-
ported and in 1987-1994,474. In 1987-1994,
220 deaths were associated with use of ionic
HOCM alone, 32 with ionic LOCM alone, and
214 with nonionic LOCM alone. The calcu-
lated occurrence of 1 death per 200,000 or
more examinations was comparable with pub-
lished data. The authors remarked that the
Radiopaques
availability of LOCM in the United States did
not lead to a marked decrease in the contrast
media-related deaths.
Lasser et al. (543) compared the patient re-
actions to ionic and nonionic contrast media
based on data on reactions to X-ray contrast
materials collected under the supervision of
the FDA Division of Epidemiology and Sur-
veillance and manufacturer data from 1990
through 1994. In 1992-1994 there were more
than 16 million examinations performed in
toto each year in angiography, CT, and urog-
raphy. The ionic HOCM, nonionic LOCM (io-
hexol, iopamidol, and ioversol), and ionic
LOCM (ioxaglate) had the following inci-
dences per million intravascular examinations
for total reactions 193.77:44.44:142.52; for se-
vere reactions, 37.44:10.52:33.61; and for
deaths, 3.90:2.07:6.39, respectively. The inci-
dence of severe reations to total reactions was
higher with nonionic LOCM (23.7%)and ioxa-
glate (23.6%)than with ionic HOCM (19.3%).
The incidence of death to severe reactions was
19.7% with nonionic media, 19.0% with ioxa-
glate, and 10.4% with ionic media. In patients
with pathologic cardiac conditions, the inci-
dence of renal failure was 3.6 times higher
with LOCM than with HOCM.
Bettmann et al. (544) created a prospective
registry for surveillance of incidence of Sd-
verse events related to ionic and nonionic con-
test media in angiography, to identify patients
likely to benefit from use of low osmolality
contrast agents (LOCAs) and to ascertain risk
factors for increased incidence of an adverse
event. The registry consisted of data of 75,616
studies from 60,891 patients in angiography at
26 high volume institutions, 56% with non-
ionic LOCAs, 8% with the ionic LOCA, and
36% with HOCAs in a 2-year study (July 1,
1990 to June 30, 1992). Most major risk fac-
tors correlated with an increased incidence of
adverse events, but varied with the type of
procedure, with a higher incidence associated
with cardiac and interventional procedures.
The incidence of adverse events was signifi-
cantly increased with use of HOCAs, but se-
vere adverse events were not much different
between ionic and nonionic contrast agents
except for cardiac procedures. Patients with a
previous reaction or more than one other ma-
6 Organic Iodine Compounds
jor risk factors are most likely to benefit from
use of LOCAs.
Several hypotheses were postulated on the
pathogenesis of contrast mediareactions. Lalli
(545),after reviewing a large number of severe
reactions to contrast media and mortality in
cholangiography, angiography, and urogra-
phy, proposed that all reactions are based on
the effect of contrast media on the central ner-
vous system. Contrast media are 400-1400
times more toxic intracisternally than intra-
venously (213,546). Penetration of the blood-
brain barrier and direct stimulation of the
brain by contrast media can produce abrupt
responses leading to immediate and severe
contrast media actions. Lasser (508, 547)
noted that the nonionic contrast media have
substantially reduced the local toxicity in ani-
mal and human studies, although the impact
on systemic toxicity is less clear. The author
postulated an allergy diathesis to prime a pa-
tient for a contrast media reaction and be-
lieved that bradykinin may contribute signifi-
cantly to the reactions. Contrast media
disrupt endothelium surfaces, causing de-
granulation of mast cells, releasing histamine
and substances capable of contact activation
(548).These substances are listed as an endog-
enous heparin-like material (EHM), a cryptic
soluble surface (CSS), or a negative surface
(549) and a complex of the plasma inhibitor
a,-macroglobulin with kallikrein (508), that
can cause complement activation to release
the Hageman factor XII, inducing sequen-
tially the cleavage of prekallikrein to produce
kallikrein and the cleavage of kininogen to
produce bradykinin. Bradykinin is a more per-
sistent and .wtent mediator than histamine in
anaphylaxis and may mobilize arachidonic acid
to produce leukotrienes and vasoactive prosta-
glandins. Contrast media inhibit angiotensin-
converting enzyme, responsible mainly for in
vivo bradykinin hydrolysis. Premedication with
corticosteroids may reduce the concentration of
complement activators. Lasser et al. (512, 549)
examined and evaluated contact system dynam-
ics in patients' plasma and compared the plas-
mas of asthmatic patients with that of the con-
trols. Moreau et al. (550) discussed Lalli and
Lasser's theories and showed the biolo&al - fects the red blood cell membrane and trans-
chain reactions induced by contrast media and
those blocked by premedication with drugs.
Brasch (541, 552) reviewed the severe, im-
mediate reactions to contrast media and sug-
gested that reactions, often designated as ana-
phylactoid, may have an antibody basis. A
prospective study by Laroche et al. (543) on
the mechanisms of severe, immediate reac-
tions to iodinated contrast material gave sup-
port to this theory. Laroche et al. measured
the levels of plasma histamine, tryptase, and
urinary methylhistidine by radioimmunoas-
say or immunoradiometric assay in blood and
urine samples from controls and from patients
who experienced allergic-type reactions after
the administration of contrast material. The
serum level of specific immunoglobulin E
(IgE) against ioxitalmate or ioxaglate was de-
termined by the radioactive anti-IgE antibody
techniques of Gu6ant et al. (544). The human
anaphylatoxins C3a and C4a were measured
in plasma using radioimmunoassays, and se-
rum C3 and C4 levels measured by nephelom-
etry. The patients with severe reactions all
showed higher measured values than those of
the controls, and the values increased with se-
verity of reactions. Histamine concentration
decreased rapidly with time and reached nor-
mal within 1 h after the reaction, but the
tryptase level decreased more slowly than his-
tamine. The half-life of histamine in healthy
volunteers is less than 2 min. Tryptase and
histamine levels are useful for the diagnosis of '
anaphylactoid reactions during anesthesia.
The study showed that the frequency of imme-
diate reaction is higher with ionic contrast ma-
terial of high or low osmolality than with non-
ionic contrast material, and that patients with
risk factor of a previous reaction to ionic con-
trast material react less frequently to nonionic
contrast material. The study pointed out the
involvement of IgE-mediated reactions.
6.5.4 Toxic Effects on Red Blood Cells.
Contrast media produce morphological changes
of red blood cells by altering their size and
shape and also increase their tendencies for
aggregation (555, 556). The change in mor-
phology of red blood cells is caused by chemo-
toxicity and hyperosmolality of the contrast
medium (557, 558). The chemotoxic effect af-
forms the normally biconcave disc-configured
red blood cell into a crenated cell known as an
548
echinocyte. Ionic and nonionic contrast media,
such as iohexol, iopentol, metrizamide, and
metrizoate in solution isotonic to blood pro-
duce echinocytes, but high concentrations of
iopamidol, iohexol, and iopentol also produce
stomatocytes. Nonionic dimer iodixanol trans-
forms the normal red blood cells entirely to
stomatocytes. These two types of cells differ
from each other in relation to the sites of con-
trast medium binding on the bilayer cell mem-
brane. The exact mechanism is unclear, but
shift from echinocytes to stomatocytes may oc-
cur by transbilayer diffusion, a well-known
phenomenon in drug-erythrocyte interaction
(557, 559). In addition to the chemotoxic ef-
fect, the hyperosmolality of the contrast me-
dium also affects the morphology of the red
blood cells by pulling water, as it were, out of
the normal cells to produce a shrunken red
blood cell, known as a desicocyte. Among the
nonionic contrast media that cause echinocyte
formation, metrizamide shows the most che-
motoxic effect. Iohexol, iopamidol, and iopen-
to1 are comparable to one another and show
the least chemotoxic effect on cell membrane
(557,558). Bucherer et al. (560) used a micro-
manipulation technique to visualize the defor-
mation of individual erythrocytes and found
that the low osmolar monoacidic contrast
agent ioxaglate makes the erythrocyte mem-
brane less rigid and the nonionic iopamidol
makes the membrane more rigid compared to
the control. Hardeman et al. (427) compared
the in vitro effects of ioxaglate, iohexol, and
iopamidol in different iodine concentrations
on whole blood, collected from healthy adults
and uremic patients, with respect to RBC mor-
phology and aggregation and found that RBCs
in the presence of 50% iohexol and iopamidol
were considerably rigidified, whereas the ad-
dition of 50% ioxaglate resulted in only mod-
erate RBC rigidification. Iohexol gave a mix-
ture of normal biconcave erythrocytes and
echinocytes, iopamidol mostly echinocytes,
and ioxaglate mostly normal RBCs with a high
degree of rouleaux formation in all different
samples tested. Parvez and Pate1 (561) ob-
served by transmission electron microscopy
that ioxilan affected the erythrocyte mem-
brane less than iohexol and iopamidol; the lat-
ter two produced acanthocytes, whereas iox-
ilan had no effect on erythrocyte morphology.
Radiopaques
Human erythrocytes at rest have a diame-
ter of approximately 7.5 pm with a higher de-
gree of deformability (558, 562). The normal
disc-shaped erythrocytes are aggregated in
rouleaux formation and retain a smooth sur-
face, allowing the cells to bend and deform to
pass through capillaries with diameters rang-
ing from 3 to 5 pm (563). A high percentage of
crenated and deformed cells inhibits rouleaux
formation and causes disaggregation. Normal
red blood cells have an average water content
of 66.46 + 1.12% by weight and may lose up to
11% of internal water on coming into contact
with a hyperosmolar solution of contrast
medium (564). The loss of internal water in-
creases the red blood cell viscosity, which re-
sults in reduced deformability and rigidifica-
tion of red blood cells, causing increased
resistance to flow and disturbance of the rhe-
ology of blood flow in microcirculation (565,
566). The rigidified erythrocytes can block the
pulmonary capillaries and cause an increase in
pulmonary arterial pressure. The high viscos-
ity of the contrast medium may prolong its
passage through the vessels compared to that
of less viscous solutions. Increased contact of
contrast medium with the vessel wall can
cause endothelial cell contraction, leading to
endothelial damage and patches of denuda-
tion, which can be the location of thrombus
formation (567).
.
Nonionic contrast media, although more
biotolerant than ionic contrast media, pose a
serious risk of thromboembolism during clin-
ical cardiac procedures. A critical review
shows that both ionic and nonionic contrast
media prolong whole blood and plasma clot-
ting times in a dose-dependent manner and
inhibit in vitro aggregation of platelets, with
the nonionic contrast media exerting less in-
terference in coagulation and platelet aggre-
gation than the ionic contrast media (568).
Kurisu and Tada (569) show that in a canine
model, the ionic low osmolar ioxaglate has a
greater anticoagulant effect than that of the
nonionic contrast agent iopamidol, which is
about the same as the saline control in its in-
hibition of clot formation. Levi et al. (570), by
use of 1251-labeledfibrinogen as a marker to
study the effect of contrast media on throm-
bus (aggregated platelets) growth in a rabbit
jugular vein thrombosis model, showed that
6 Organic Iodine Compounds
the ionic ioxitalamate and ioxaglate exhibit
marked inhibition of thrombus growth,
whereas the nonionic contrast agents as a
group show prothrombotic effect with iopam-
idol promoting more thrombus growth than
iopromide and iohexol. The thrombogenic ef-
fect of iopamidol can be inhibited by simulta-
neous administration of 80 IU/kg heparin. Ac-
cording to Hwang et al. (5711, the optimal
heparin concentration to achieve a complete
anticoagulant effect is 5 IUImL.
Thrombus formation is a part of the coag-
ulation cascade, complicated and involving
many factors, and occurs when a nonionic con-
trast medium is mixed with whole blood (572,
573). According to Ing (574), ionic and non-
ionic contrast media exert their actions at dif-
ferent stages of the clotting process. Ionic con-
trast media inhibit thrombin activity and
fibrin polymerization, whereas nonionic con-
trast media inhibit only fibrin polymerization.
Ioxaglate inhibits both the coagulation se-
quences before thrombin generation and the
final stages of fibrin monomer formation and
polymerization, whereas the nonionic iopam-
idol and iohexol inhibit coagulation primarily
after the generation of thrombin. This differ-
ence in inhibition may lead to serious compli-
cations for nonionic contrast media because
some unclotted mixtures of blood and non-
ionic contrast agents, which may contain
thrombin, may cause clotting on reinjection.
Grabowski et al. (575) showed that both non-
ionic and ionic contrast media retard clotting
in plastic and glass angiography syringes in
comparison to saline control, and ionic con-
trast agents are stronger anticoagulants. Con-
tainer and syringe surfaces influence the for-
mation of thrombus in mixtures of nonionic
contrast media and blood, and plastic syringes
were found to retard clotting better than glass
ones (573, 575). By use of scanning electron
microscopy Corot et al. (576) investigated the
surfaces of polyethylene (PE) and polyure-
thane (PU) and inside surfaces of catheters
after contact with mixtures of blood and con-
trast media, such as diatrizoate, ioxitalamate,
ioxaglate, iopamidol, and iohexol. The PE sur-
face was relatively heterogeneous and the PU
surface was smooth and homogeneous. Iopam-
idol and iohexol, when mixed with the blood,
formed thick fibrin fibers on the PE surface
and not on the smooth PU surface. In compar-
e
ison, the ionic contrast agents showed no sig-
nificant fibrin formation on either of these
surfaces. The blood-catheter interface at the
internal surface of catheters after injection of
ioxaglate or iohexol or iopamidol, collected
from different European clotting centers,
showed signs of blood activation, platelet acti-
vation, fibrin formation, or occurrence of red
thrombi. These findings show that the clotting
process is procedure dependent and varies be-
tween these centers and that ioxaglate is sig-
nificantly- better than the low osmolality- non-
ionic contrast media in retarding clotting.
Krause and Press (577)studied the effect of
nine contrast media and four gadolinium che-
lates on blood coagulation and measured their
thromboplastin time (TPT). All contrast me-
dia influence TPT, but the driving force is
ionic charge. The authors found that these
agents induced a stastistically significant in-
crease in TPT, listed in the following ascend-
ing order of the increase: plasma = saline <
gadobutrol < iodixanol = iotrolan = iohexol
(different from iodixanol) = iopental (differ-
ent from iodixanol and iotrolan) < iopromide
< iomeprol << Gd-DTPA << ioxaglate << dia-
-
trizoate. Ionic contrast agents interact with
the coagulation cascade to a greater degree
than nonionic contrast agents and are able to '
influence TPT and to combine with calcium
ions. The TPT in human vlasma was found
to be longer than that in other species, and
the interspecies ranking for TPT was dog <
rabbit < rat < human. The addition of hepa-
rin to contrast media significantly prolonged
TPT.
In a human endothelial cell culture, ioxa-
glate appears to be more potentially toxic to
endothelium than diatrizoate, iohexol, iopro-
mide, ioversol, iotrolan, and gadolinium-
DTPA (578). Nitric oxide, known earlier as
"endothelial-derived relaxing factor," may
also play a role in mediating the chemotoxicity
of contrast media, given that infusion of
iothalamate accompanied by L-arginine ana-
logs could protect the rats and raise the com-
pletely lethal dose (LD,,,) significantly (579).
Wiesel et al. (580) observed that slight but
significant platelet activation and thrombin
generation in patients during coronarography
occurs with both ionic and nonionic contrast
550
media, and that both types are potentially
thrombogenic and behave as activators of
platelet and blood coagulation. These authors
observed only signs of biological activation of
hemostasis without clinical symptoms. In pa-
tients with atherosclerotic lesions, contrast
media may interfere with homeostasis by fa-
cilitating platelet adhesion. The risk factors
for nonionic contrast media-induced thrombo-
sis are listed (538). Koza et al. (581) evaluated
platelet activation by ionic and nonionic con-
trast media in native heparinized or hirudi-
nized human blood by use of flow cytometric
analysis techniques, measurement of platelet
factor 4 and thromboxane B,, and antibodies
to identify platelets (CD-61) and activated
platelets (CD-62), to establish that dose- and
time-dependent platelet activation was ob-
served in nonionic, but not in ionic contrast
media. The study suggests that contrast me-
dia use is associated with significant levels of
platelet activation and that the anticoagulant
heparin or combinant hirudin (where heparin
is contraindicated) is preferable for use with
nonionic contrast media to reduce contrast
media-induced platelet activation.
6.5.5 Cardiovascular Effects. To visualize
blood vessels and other structures of small
thickness, a sufficient radiopaque dose must
be used. Contrast media for angiography are
usually administered intravascularly in high
concentrations and large volumes (582). Dur-
ing coronary arteriography, the blood is re-
placed for a short period of time with a con-
trast medium solution (583).The effects that a
contrast medium exerts on the central cardio-
vascular function will be determined by the
iodine concentration, osmolality, viscosity,
and the rate of injection. In angiocardiography
the contrast medium can cause (1) electro-
physiologic effect, such as heart rate alter-
ations, atrioventricular conduction delay, and
ventricular arrhythmias; (2)hemodynamic ef-
fect, such as hypotension, inhibition of left
ventricular coronary contractile site, and al-
terations in coronary blood flow; and (3) met-
abolic effects, such as hypocalcemia and alter-
ations in myocardial metabolism (469, 584).
Contrast media can cause fluid shifts upon
mixing with blood, "pulling" out water from
the red blood cells, and blood vessel endothe-
Radiopaques
l i d cells within a few milliseconds of exposure.
The mixture of blood and contrast medium,
when entering and passing through the lungs,
can cause changes in pulmonary pressure
(582,585). In addition, contrast media can ex-
ert a direct effect on the myocardium, depress-
ing myocardial contractility (584). Thus, sys-
temic hypotension, changes in the activity of
the smooth muscle cells in vessel walls, aggre-
gation and crenation of red blood cells, and
hypervolemia are examples of vascular reac-
tions induced by the administration of con-
trast media. Agglutination and crenation of
red blood cells cause blood sludging and re-
duce the cells' ability to undergo deformation,
thus blocking the blood flow to capillaries and
leading to pulmonary hypertension and re-
lated physiological responses (495-498). The
hypervolemia resulting from a shift of water
elevates the ventricular filling pressure and
increases the cardiac output from increased
ventricular stroke work. The potassium ions
released from shrinking and crenating red
blood cells contribute to the lowering of sys-
temic vascular resistance and blood pressure
(499).
Sheu et al. (586) compared the effects of
ionic monomer diatrizoate and nonionic
monomer iohexol on coronary hemodynamics
and myocardial metabolism. In dog hearts, the
intracoronary injection of iohexol produced an
increase in myocardial contractibility, aortic
systolic pressure, and myocardial oxygen con-
sumption. In contrast, the injection of dia-
trizoate produced a decrease in these parame-
ters. This difference between ionic and
nonionic contrast media was significant. Nev-
ertheless, the degree of increased coronary
blood flow after coronary injection was similar
for both agents.
In coronary arteriography, ionic mono-
mers, because of their high osmolality and
binding to calcium and magnesium in plasma,
elicit more physiologic effects than nonionic
monomers. The nonionic molecules do not
combine calcium; the chelators used in the for-
mulation have less calcium binding capacity;
and the osmolality of nonionic monomers is
only half that of the ionic monomers. Thus,
nonionic contrast media cause fewer fluid
shifts, less vasodilation, arrhythmia and fibril-
lation, and minimal cardiac depression. In an-
6 Organic Iodine Compounds
imal experimentation, injections of short du-
ration (2-5 s) produce fewer incidences of
myocardial fibrillation than prolonged injec-
tions (25 s) (482). The nonionic dimers or bis
compounds, such as iotrolan and iodixanol,
further reduce the clinical adverse reactions
compared to those of the nonionic monomers.
The dimers are isotonic to blood but have vis-
cosities (>10 mPa.s) more than double the vis-
cosity of blood (4 m P a 4 and more than 8
times the viscosity of plasma (1.2 mPa.s), with
no apparent adverse effects (582). Ventricular
fibrillation and serious arrhythmia have been
linked to hyperosmolality and the absence of
sodium ions. The safety of iotrolan is related to
its isotonicity and the small amount of sodium
ions it contains (482).
The safety, tolerance, and diagnostic effi-
cacy of iopentol, iopromide, iohexol, ioversol,
and iopamidol have been compared in different
cardiac angiographicprocedures (587490).In a
canine heart model, ioversol with added so-
dium in the form of NaCl can reduce the pro-
pensity for spontaneous ventricular fibrilla-
tion and arrhythmias but it also causes an
increase in the QT interval in the electrocar-
diogram. The QT interval is associated with
cardiac conduction and depolarization and re-
polarization. The prolongation of the QT in-
terval, unlike that caused by ionic contrast
media, is not an indication of arrhythmogenic-
itv- (591). The influence of sodium on electro-
cardiography during coronary angiography
with iopromide has also been investigated
(592).
Other factors may exhibit synergism in re-
actions with contrast media. In mice, contrast
media with cardiac glycoside cause higher
mortality than contrast media alone (593). In-
compatibility of contrast media with other
pharmacological agents has also been noted
(594). Anaphylactoid reactions from contrast
media can also be associated with P-blocker
exposure, cardiovascular disorders, or asthma
(595).
Muschick et al. (596) compared the hemo-
dynamic
" effects of 11 iodinated contrast me-
dia, including ionic (diatrizoate, ioxaglate),
nonionic monomers (iohexol, iopromide, io-
pamidol, iopentol, ioversal, iomeprol), and
nonionic dimers (iotrolan. iodixanol) in rats.
The animals were injected with contrast me-
dium at a high dose of 1.2 g iodinekg into the
left ventricle. Transient changes in blood pres-
sure (systolic and diastolic), heart rate, cardiac
output, left ventricular end diastolic pressure,
total peripheral resistance, and electrocardio-
gram within a few minutes post-injection were
observed. In a healthy rat, the authors noted,
all contrast media tested are safe but generate
a few side effects. Low osmolar nonionic mo-
nomeric contrast media have a clear benefit
over high osmolar contrast media. Nonionic
isotonic dimers are best, with minimal side ef-
fects, and are likely to provide a lower risk if
used in ventriculography and coronary an-
giography.
6.5.6 Nephrotoxicity. In urography and
arteriography more than 90% of the injected
dose of contrast media is excreted through the
kidney (597). Contrast media adversely affect
the excretory function of the kidneys, partly
by osmolality and partly by chemotoxicity. Di-
uresis, changes of renal blood flow, osmotic
nephroses, albuminuria, enzymuria, and
changes of glomerular filtration rate are re-
ported in animal studies of contrast media-
induced nephrotoxicity (598). In patients with
normal and abnormal kidneys, contrast media
can cause acute deterioration in renal func-
tion, such as reduced glomerular filtration
'
rate, medullary necrosis, and transient pro-
teinuria of glomerular origin, associated with
the increased elimination of &-microglobulin
(599, 600). Individuals with preexisting renal
insufficiency, diabetes mellitus, dehydration,
cardiovascular disease, advanced age, multi-
ple myeloma, hypertension, hyperuricemia,
and several contrast examinations within 24 h
have a high-risk of kidney malfunction (598,
601). Low osmolality monomers and dimers,
such as iohexol, iopamidol, iopromide, iover-
sol, ioxaglate, and iotrolan, are less nephro-
toxic in high risk patients than high osmolal-
ity ratio 1.5 agents, such as diatrizoate, for
example (602, 603, 607, 608). The severity of
the nephrotoxic effects is determined by the
dose and rate of injection (463, 609, 610). In
the clinical setting patients with normal renal
functions respond similarly to ionic and non-
ionic contrast media (611-614), and the renal
effects are transient only in these patients
(600).

Contrast media cause damage to the vascu-
lar, glomerular, and tubular structures of the
kidneys and changes in urine profiles, which
may lead to acute renal failure (463,597,615,
616). In the canine model, renal ischemia po-
tentiates nephrotoxicity (603). Deray et al.
(604)studied renal hemodynamics in a normal
and ischemic dog kidney and found that io-
dixanol induced a greater decrease in renal
blood flow than did ioxaglate and produced a
marked intrarenal vasoconstriction. Rauch et
al. (605) compared the effects of contrast me-
dia (diatrizoate, iohexol, and iopamidol) on re-
nal vasoconstriction in human, rabbit, dog,
and pig arteries and found that all contrast
media elicited dose-dependent reversible con-
tractions in human, rabbit, and dog but not in
pig renal arteries. All the contrast media
tested exerted a vasorelaxing effect on pig
renal artery. Nygren et al. (606) found that
intravenously administered ioxaglate and io-
pamidol enhance the microvascular obstruc-
tion, as a consequence of red-cell trapping
evoked by ischemic injury, and that iopamidol
may induce local impairment in renal medul-
lary microcirculation in a normal kidney.
The response of renal vasculature to con-
trast media exposure is biphasic. Immediately
after intraarterial or intravenous injection of
the contrast medium peripheral resistance de-
creases (vasodilation) and renal blood flow in-
creases, followed by renal vasoconstriction
and decreased renal blood flow. The size of the
kidney changes and reaches its maximum size
within the first 5 min after injection (598). The
contrast media within the renal vascular bed
attract water from the interstitial space and
possibly from the renal tubules. In this renal
hemodynamic and fluid shift, contrast media
and some low molecular weight substances are
freely filtered by the glomeruli and remain in
the tubular lumen in a high concentration for
a relatively long time, causing tubular damage
(609). Water, contrast media, and urinary
components that are not reabsorbed in the tu-
bules are excreted as hyperosmolar urine in
diuresis (597,609, 615).
Contrast media cause damage to the endo-
thelia of renal vasculature and release vasoac-
tive peptides, such as renin, prostaglandins,
and kallikreins. Other associated events are
deformation and rigidification of red blood
Radiopaques
cells and platelet accumulation in the areas of
denuded blood vessels (598). The relation of
the vasoactive peptides and associated events
to vascular hemodynamic effects is not well
understood.
Urographic and uroangiographic contrast
media cause glomerular damage by altering
membrane permeability, thus allowing pro-
tein leakage into the urine, resulting in pro-
teinuria. After renal angiography, protein ex-
cretion in the urine may increase by a factor of
more than 1000 (598, 617). In normal rats,
albumin levels return to normal after 2 h
(616). Massive proteinuria from glomerular
damage may cause protein precipitation in tu-
bular lumen, decreased urine flow, and may
produce acute renal failure (463). It has been
shown that nonionic iohexol produces less, if
any, albuminuria in animals and humans than
ionic contrast media or metrizamide (598). Io-
hexol has been proposed for use as a marker
for glomerular filtration rate (618, 619).
One of the nephrotoxic effects of contrast
media is the vacuolization of the proximal tu-
bular cells (598). Iohexol and iotrolan induce
more significant and longer-lasting vacuoliza-
tion than the ionic diatrizoate and nonionic
monomer iopromide (620). Iohexol also causes
significantly more tubular vacuolization and
capillary congestion than another nonionic
contrast agent, iobitridol(301). This vacuolar '
response reflects lysosomal changes and the
chemotoxicity of contrast molecules. Tubular
vacuolization is also known as osmotic ne-
phrosis. Wasaki et al. (621) compared the toxic
effects of iobitridol and iohexol on the acute
renal failure (ARF) kidney in a rat model and
found that iobitridol produced a lower degree
and incidence of renal tubular injury of renal
proximal and distal tubules compared with io-
hexol. In the rat renal slice system, iobitridol
had significantly less effect on thep-aminohip-
puric acid accumulation compared with that of
iohexol. There were no changes of intracellu-
.
lar ~otassiumcontent.
Tubular damage causes enzymuria. Uri-
nary excretion of the lysosomal enzyme
N-acetyl-p-D-glucosidase(NAG), the cytoplas-
mic enzyme lactic dehydrogenase (LDH), and
the brush-border enzyme gamma glutamyl
transpeptidase (GGT),caused by contrast me-
dia, has been studied in rats with drug-in-
6 Organic Iodine Compounds
duced nephropathies (622, 623). These en-
zymes with molecular weights greater than
70,000 are not filtered by the glomerulus
(624). The level of enzymuria appears to cor-
relate with the degree of proximal tubular cell
injury. This may suggest that the urinary en-
zyme levels might be a more sensitive param-
eter than the serum creatinine levels to assess
the renal toxicity of iodinated contrast media
(616,625,626).
Ed6e and Bonnemain (627) reviewed the
reliability of experimental models, from meth-
odological considerations to pathophysiology,
of iodinated contrast media-induced acute re-
nal failure and nephropathy. Deray and Ja-
cobs (628, 629) reviewed radiocontrast neph-
rotoxicity and renal tolerance of nonionic
dimers. Nonionic dimers (iotrolan and iodixa-
nol) are iso-osmotic and have high viscosities.
Iotrolan at 300 mg I/mL has a viscosity of 17.8
mPa-s at 20°C and at 320 mg IImL, 24.6 mPa-s.
In comparison, iobitridol at 300 mg I/mL has a
viscosity of 11.2 mPa-s at 20°C. These chemi-
cal characteristics that cause an increase of
the reflection coefficient, characterized as "the
tendency of solutes to be retained in the vas-
culature with resulting prolongation of the
phase of osmotic expansion of plasma vol-
ume," may be responsible for the toxic symp-
toms. Vergare and Sequel (630) recommended
warming the contrast media to 35°C before
injection to improve tolerance.
6.5.7 Neurotoxicity. Contrast agents are
toxic to the central nervous system (631).
Their presence there is brought about either
by direct injection into the cerebrospinal fluid
or by leakage through the blood-brain barrier.
Contrast agents are more toxic when adminis-
tered intracisternally than intravenously
(214). The ratio between intravenous and in-
tracisternal LD,, values in the rabbit can be as
high as 1450 to 1 for diatrizoate sodium (213,
546). Adverse effects caused by ionicity, che-
motoxicity, and hyperosmolality of contrast
media can range from discomfort to the
patient to the disruption of the blood-brain
barrier (BBB), epileptogenicity, and late
arachnoiditis.
The transport of materials from the blood
to the tissues (or extravasation) in the brain,
unlike that in all other organs, is restricted by
the BBB (632). Contrast media administered
by intrathecal and intracisternal injections
will bypass the BBB and come in direct contact
with the neurons and glial cells (633). The
BBB refers to the tight junctions between the
cerebrovascular endothelial cells in cerebral
blood vessels, which form a physical barrier to
the exchange of ions and large molecules be-
tween blood and brain (634-636). Concen-
trated solutions of electrolytes and nonelec-
trolytes such as urea can reversibly open the
barrier by separating the tight junctions
through osmotically shrinking the endothelial
cells (637-640). Barrier opening may be eval-
uated by the entry of dyes (e.g., Trypan blue,
Evans blue) (634,641) or radioactive ions (e.g.,
""Tc, 32P)(642) into the brain parenchyma.
The percentage penetration of 1311-tagged
iothalamate and diatrizoate in the rat cerebral
cortex, studied by use of historadioauto-
graphic techniques, correlates well with the
degree of severity of neurotoxicity (643).
When there was little or no penetration of con-
trast medium into the brain tissue, no convul-
sions were observed. These experimental
methods when applied together with those of
induced convulsions (641-643)and damage to
tissue cultures of neurons (644), to determine
the neurotoxicity of contrast agents, may or
may not necessarily measure the same molec-
ular property responsible for neurotoxicity. In
fact, cytochemical studies show that the BBB
damage induced by contrast medium differs
from the BBB damage caused by other types of
insult, in that the plasma membrane-bound
enzyme Ca2+-ATPaseand the negative charge
that resides on the endothelial luminal surface
are not affected (645). Damage from different
contrast media may also differ. For example,
the nonionic dimer iotrolan causes more BBB
damage in the basal ganglia than in the cortex,
whereas the reverse is true for the ionic dia-
trizoate (646). To predict the entire spectrum
of neurotoxicity of contrast media, Sovak et al.
(647) proposed the use of a number of methods
for assessment of neurotoxicity in testing new
nonionic contrast media.
The neurotoxicity of contrast media is de-
termined by their lipid solubility or partition
coefficient (631). Lipid solubility regulates the
passage of these agents through the unaltered
BBB. The partition coefficients of iothalamate,
554
ioxitalamate, diatrizoate, metrizoate, iodam-
ide, diprotrizoate, and 2,4,6-triiodobenzoate
have been calculated (631), using Hansch's
rule for additive constitutive properties (648-
651). This study shows that the neurotoxicity
of these agents increases with increase in the
combined partition coefficient P and is related
to the brain uptake index. The relation is sim-
ilar to that observed by Oldendorf (652) be-
tween the chain length of short-chain mono-
carboxylic acids and their transport across the
blood-brain barrier, which reaffirms the de-
pendency of neurotoxicity on lipid solubility.
Nonionic contrast molecules designed with
4-6 hydroxyl groups in the molecule to maxi-
mize hydrophilicity have been shown to have a
good correlation between partition coefficient
(octanoVwater) and neurotoxicity (653). For
example, the hydrophilicity increases as the
partition coefficient decreases, in the case of
four monomeric nonionic contrast agents ac-
cording to the ranking: metrizamide > iop-
amidol > iohexol > ioversol (654, 655).
Their neurotoxicity, measured in terms of
lethality, EEG effects, and motor effects, de-
creases proportionately with the increase in
hydrophilicity.
In addition to osmolality and lipid solubil-
ity, the cations, anions, and chemotoxicity of
contrast media also contribute to neurotoxic-
ity. The sodium salts of diatrizoate and
iothalamate are about three times more neu-
rotoxic than the meglumine salts when com-
pared at comparable doses in rats by intracis-
ternal application, and the diatrizoate anion is
about 10 times more neurotoxic than the
iothalamate anion (656). In both hypertonic
and hypotonic ranges, neurotoxicity increases
with increasing concentration of contrast me-
dium. When comparing the neurotoxicity, not
only the mold concentration of the contrast
medium but also the brain weight of the exper-
imental animals should be considered. Evil1 et
al. (657) compared the chemotoxic effects of
nonionic contrast monomers iohexol and io-
verso1 and dimers iodixanol and iotrolan in the
rabbit using 99mTc-pertechnetateas a marker
for BBB damage and mannitol as osmolality
control. At comparable osmolalities, mannitol
causes no brain 99mTcuptake, whereas the
contrast agents cause significant ""Tc up-
take according to the order: iohexol > iodixa-
Radiopaques
no1 > ioversol > iotrolan, with iohexol causing
more BBB damage than iotrolan. Motoji et al.
(658)studied the neurotoxicity of contrast me-
dia using [8-l4C1dopamine as a marker for
BBB damage in Monogolian gerbils and found
that both meglumine sodium amidotrizoate
and iohexol caused damage to the BBB, as
manifested in cerebral cortex, brain stem, and
hippocampus sections in the left hemisphere.
Iopamidol caused a much smaller but signifi-
cant effect, and ioversol caused no damage to
the BBB. Michelet (659)used iothalamate and
iohexol as reference contrast agents and
99mTc-DTPA,lZ5I-labeledhuman serum albu-
min and Trypan blue as tracer to evaluate de-
grees of damage to the BBB in male albino
rabbits. The ionic monomer iothalamate
caused marked BBB damage, based on a large
extravasation of tracers, the nonionic mono-
mers iopentol and iohexol caused some BBB
damage, and the dimer iodixanol caused only
minor changes to the barrier. The author rec-
ommends iodixanol as a safe contrast agent for
computed tomography of the brain. Harnish
et al. (660) found that iohexol or ioxilan at a
solution concentration of 175 mg I/mL caused
no BBB damage in anesthetized Wistar rats.
This strain of rats has been reported to be
more sensitive than other strains to the intra-
thecal effects of contrast agents (187). Whis-
son et al. (661) studied acute hypertension
(HT) as a risk factor for damage to the BBB in
carotid angiography with nonionic contrast
media (iopamidol) in groups of rats. Anesthe-
tized animals received intravenous injections
of 99mTc-pertechnetateand horseradish per-
oxidase as tracers for quantitation and histo-
logical examination. Two groups of rats re-
ceived metaraminol to raise their blood to
between 165 and 190 mmHg peak systolic and
then received intracarotid injection of saline
and iopamidol at 300 mg I/mL. Two groups of
normotensive rats received normal saline and
iopamidol as controls. The authors found that
the extravasation of both tracers were signifi-
cantly greater in the hypertensive group that
received contrast media than in the other
groups and that acute hypertension potentiates
the BBB-damaging effects of nonionic contrast
media during carotid angiography in rats.
Luzzani et al. (426) assessed the neurotol-
erability of six monomers (iomeprol, iopam-
6 Organic Iodine Compounds
idol, iohexol, iopentol, ioversol, and iopro-
mide) and three dimers (iofratol, iotrolan, and
iodixanol) of nonionic X-ray contrast media in
rats and mice by their median lethal dose
(LD,,), compared to controls. The contrast
agents were administered by intracerebroven-
tricular 6c.v.) injection to mice and by intra-
cisternal (i.c.i.) injection to rats. The iodine
contents of monomers and dimers injected
were 350-370 and 300-320 mg IImL, respec-
tively. The toxic effects for all contrast media
included convulsion, dyspnea, hypoactivity,
and sedation. In mice iohexol was significantly
more neurotoxic than other monomers, and
the dimers had similar neurotoxicity but were
more toxic than the monomers; this difference
became more evident when the concentrations
of LD,, values were expressed in mmol/kg in-
stead of mg I/mL. In rats iopentol and iopro-
mide were sufficiently more neurotoxic than
others that their LD,, values could be accu-
rately determined. The authors attributed the
observed differences in neurotoxicity of the
contrast media to differences in chemical
structures, that is, to chemotoxicity rather
than hydrophilicity or physicochemical char-
acteristics of pharmaceutical formulations of
the contrast media. Mice and rats had differ-
ent sensitivities toward contrast media, ap-
parently from drug actions targeted at differ-
ent areas in the brain as a result of different
sites of injection.
Damage to the spinal cord by contrast me-
dium also causes neurotoxicity (456,457,662).
Antoni and Lindgren (663) first observed spi-
nal cord damage after aortography. A survey
conducted by Killen and coworkers (664,665)
showed that 90% of the reported spinal cord
injuries was caused by acetrizoate, especially
at high concentrations. Others (666-670)
have shown that all water-soluble angio-
graphic agents can inflict damage on the
spinal cord. Contrast agents such as sodium
iodide, acetrizoate, iodomethamate, dipro-
trizoate, diodone, diatrizoate, and thorium ox-
ide have neurotoxicity decreasing in that or-
der (670). Foster et al. (671) found that
diodone, acetrizoate, and diprotrizoate have
higher toxic effects on kidney and spinal cord
after aortography than thorium dioxide, dia-
trizoate, and iothalamate. Damage to the spi-
nal cord is highly likely in any procedure that
concentrates the contrast agent in this region.
Metrizamide was the first-generation non-
ionic contrast agent for the central nervous
system found to be almost free of neurotoxic
effects (672). Metrizamide is often used as a
standard of comparison in animal experimen-
tation when newer nonionic contrast agents
are evaluated for neurotoxic effects. Adams et
al. (673) used electroencephalography (EEG)
recording to detect neurotoxic effects of con-
trast media after i.c.i. administration in the
rat. These authors found that ionic meglu-
mine iothalamate produces profound seizure-
like patterns in EEG at 30 mg IImL. In com-
parison, metrizamide produced minimal EEG
abnormalities at this concentration. Ioversol
and ioglunide produced mild to slight EEG ab-
normalities in doses ranging from 60 to 240
mg I/mL. Ioglunide was well tolerated in total
spinal column myelography in a group of 86
patients but was considered to be at a compet-
itive disadvantage in manufacture when com-
pared to iohexol and iopamidol, because io-
glunide contains a sugar moiety and is as
unstable in solution as metrizamide (673). Io-
parnidol provoked seizure-like patterns in EEG
at a considerably lower dose than metrizamide
when injected as highly concentrated solutions
(400 mg VmL) into the subarachnoid space of
the anterior fossae of guinea pigs (674).
Nonionic contrast media in either hyper-
tonic or hypotonic solution, when injected in-
tracerebrally or into the subarachnoid space of
rats, will cause distinct depression of the cen-
tral nervous system (CNS) and associated
brain functions but no excitation (675). Such
depressive action can obscure the excitatory
action caused by ionicity or chemotoxicity of
myelographic agent. When an isotonic con-
trast medium is injected and mixed with the
normally produced cerebrospinal fluid (CSF)
in the subarachnoid space of the rat, the re-
sultant mixture is hypertonic with higher lev-
els of sodium and chloride. This movement of
sodium and chloride into CSF without accom-
paniment of water is unexpected. Mennini et
al. (672) noted that contrast enhancement of
brain parenchyma is never achieved by direct
intracarotid or intravenous injection of non-
ionic contrast media, unless the BBB is spon-
taneously or experimentally broken (646).
556
This may suggest specific sites for neurotoxic-
ity other than just BBB disruption. In search
of such specific receptors, Mennini et al. (672)
found that iohexol and iopamidol at concen-
trations as high as 100 pM did not displace the
tritium-labeled ligands from their neurotrans-
mitter binding sites and suggested that the
cell responses may be mediated by the phos-
phatidylinositol second-messenger system
(676). According to Ekholm (633) and Mari-
netti (677) nonionic contrast media (iohexol,
iopamidol, metrizamide, iodixanol, and iotro-
lan) can influence the uptake of 45Caand the
incorporation of [32P]phosphateinto phospho-
lipids (PA), phosphatidylinositol (PI), phos-
phatidylinositol-4-phosphate (PIP), and phos-
phatidylinositol-4,5-biphosphate (PIP-2) in
isolated rat brain synaptosomes, but it is not
known whether these reactions are important
in clinically observed neurotoxic effects of con-
trast media.
Injection of vasodilators, vasoconstrictors,
or other agents that change the relative flow of
blood and contrast medium toward or away
from the spinal cord or alter its response to the
contrast medium can modify the neurotoxic
effects (666, 678). After intra-aortic injection,
the neurotoxicity of contrast agents may be
increased by the presence of chemicals such as
heparin (679), which increase the permeabil-
ity of the blood-brain barrier. Hypothermia,
procaine (prior-injected), 20% glucose, and
low molecular weight dextran can exert a pro-
tective effect against spinal cord damage (671,
681, 690). Diazepam can prevent clonic con-
vulsions and muscular twitchings after my-
elography with iocarmic acid (682). These side
effects can also be reduced in patients receiv-
ing levomepromazine (683). The mechanism
of these protective effects is not fully under-
stood.
6.5.8 Protein Binding. Protein binding is
reputed to affect the toxicity and selective ex-
cretion of contrast media (414,417,684). Ionic
contrast media were shown by equilibrium di-
alysis to combine with serum albumin at a
number of strong and weak binding sites on
the protein (414, 416, 417, 685). Acetrizoate,
iodipamide, and iopanoate with an open posi-
tion 5 in the substituted benzene nucleus com-
bine strongly with albumin, whereas the fully
Radiopaques
substituted triiodobenzoates such as diatrizo-
ate and iothalamate bind only weakly. Ace-
-
trizoate and iodipamide bind to bovine serum
albumin strongly at one site and weakly at
several other sites, whereas iopanoate binds
strongly to three sites on the protein. The dif-
ference in the strength of protein binding sites
may account for the species specificity of the
toxicity of acetrizoate observed in 15 species of
animals. The oral cholecystographic agent io-
phenoxic acid, which is no longer used, has a
strong aMinity of binding to plasma albumin
with a half-life in humans of about 3 years
(686). Its structure is identical to that of io-
panoate, with the exception of an OH instead
of a NH, group at position 3 of the benzene
nucleus. Iophenoxic acid is tightly bound to
. to
human serum albumin at site I as compared
iopanoate at site 11. Nonionic contrast media,
such as iohexol, iopamidol, and iodixanol, for
example, in spite of their fully substituted
benzene nucleus and high water solubility,
also interact with proteins. According to Lang
and Lasser (685, 687), the binding between
the contrast material and protein is attributed
to hydrophobic interaction.
Protein binding may affect the selective ex-
cretion by the kidney or liver of a contrast
medium (414, 688, 689). The cholegraphic
agents iodipamide and iopanoic acid show
strong binding to serum protein. Although thk
relationship between the affinity for protein
binding and the preferential uptake and excre-
tion by the liver of contrast agents has not
been clearly defined, two hypotheses have
been advanced to explain the phenomenon.
One hypothesis is that the binding with albu-
min prevents glomerular filtration of the con-
trast medium, thus providing a continuing
concentration to liver parenchymal cells (689,
6901, and the other hypothesis is based on the
free penetration of serum albumin in the
Disse's space, which allows preferential access
of the bound contrast medium to liver cells
(691). Neither of these hypotheses is tenable
because (1)it is not possible to force greater
quantities of acetrizoate into the bile by in-
creasing the serum-binding capacity of ace-
trizoate to the level of iodipamide by priming
with serum albumin (692), and (2) some sub-
stances such as Evans blue that are exten-
sively bound to serum albumin are not ex-
6 Organic Iodine Compounds
creted in the bile to any significant extent,
whereas other poorly bound compounds such
as p-aminohippuric acid are efficiently ex-
creted by the liver (414). These findings point
to the involvement of additional mechanisms
besides serum protein binding in determining
the biliary excretion of contrast medium. Glu-
curonidation can increase the bile excretion of
contrast media (693).
Sokoloff et al. (694) showed that iopanoic
acid and iodipamide, but not iothalamate,
bind to the Y and Z proteins found in the liver
and the mucosa in the small intestine. Song et
al. (695) studied the role of serum albumin in
the hepatic excretion of iodipamide and found
that the contrast agent establishes an equilib-
rium between serum protein and the intrahe-
patic anion-binding Y protein, also known as
ligandin, with the latter controlling the pro-
cess of hepatic uptake and excretion in which
serum albumin plays no role. Goldstein and
Arias (696) have studied the interaction of li-
gandin with iopanoicacid, diatrizoate, iodipam-
ide, and iodopyracet. It is apparent that the
role of ligandin in the transfer of contrast me-
dium from blood to liver is to bind the contrast
material in the liver cell for intracellular
accumulation.
Contrast media inhibit enzyme activity,
and the degree of inhibition parallels their
binding affhity to serum albumin. Iopanoate,
iodipamide, acetrizoate and isofamic acid, and
diatriazoate and iothalamate exert, in de-
scending order, an inhibitory effect on the fol-
lowing enzymes: lysozyme, glucuronidase
(two types), alcohol dehydrogenase, glucose-6-
phosphate dehydrogenase (414, 687), adeno-
sine triphosphatase, carbonic anhydrase
(6981, and cholinesterases of human red blood
cells and plasma (672,673). Cholecystographic
agents can adversely affect the glucuronida-
tion of p-nitrophenol more than urographic
agents (700).Sodium ipodate strongly inhibits
the microsomal p-nitroanisole demethylase
and aniline hydroxylase activity (701). Metriz-
amide binds reversibly with catalase (702) and
exhibits weaker inhibition of cholinesterase,
glutamic-oxalacetic transaminase, and glu-
cose-6-phosphate than that of either diatrizo-
ate or iothalamate (703).
Cholegraphic agents such as iodipamide
and iopanoic acid are about 10-100 times
more potent inhibitors of cholinesterases than
the angiographic agent diatrizoate (698).
Toxic reactions manifested as gastrointestinal
symptoms and hypertonicity of bladder from
the use of oral cholecystographic agents may
be attributed to their anticholinesterase activ-
ities. The hypotensive effects induced in ex-
perimental animals by ioglycamic acid and io-
dipamide are not completely blocked by
atropine sulfate, indicating that not all the
toxic effects of cholegraphic agents are attrib-
uted to inhibition of acetylcholinesterase ac-
tivities (704).
Contrast media induce serum complement
activation (705). The degree of activation is a
function of concentration and chemical struc-
ture of the contrast agent. The order of serum
complement activation by contrast agents is
metrizamide < iothalamate < diatrizoate <
acetrizoate < iodipamide and iopanoic acid,
which is also the order of enzyme inhibition.
Activation of serum complement results in
structural and functional alterations of cell
membranes because of the release of small ac-
tive peptides such as the anaphylatoxins,
bringing about a wide variety of reactions in-
cluding contraction of smooth muscle, in-
creased capillary permeability, release of his-
tamine from mast cells, directed attraction of
polymorphonuclear leukocytes, and release of
hydrolases from these cells. Contrast media
affect the normal process of fibrin and plasma
formation in the coagulation mechanism by
changing the fibrin fiber diameter and increas-
ing the size of protein aggregates with an al-
tered spatial arrangement of the clots (706).
Nonionic contrast media have high hydro-
philicity, extremely low toxicity, and an insig-
nificant level of protein binding of less than
3%, and may induce serum complement acti-
vation (477, 707). At a contrast medium con-
centration of 1.2 mg IImL, the extent of bind-
ing to human plasma proteins is about 1.5 ?
0.3% for iohexol, 0.1 + 2.2% for iopentol, 2.9%
for iopamidol, 4.3 + 0.3% for metrizamide,
and 7.6 2 1.5% for ioxaglate (708, 709).
Dawson (653) ranked the relative protein
binding capacity of iohexol:iopamidol:ioxag-
1ate:iothalamate as 1:1.5:3.5:4 and correlated
the partition coefficient (butanollwater)of me-
trizamide (0.37):iopromide (0.14):iopamidol
(0.ll):iohexol (0.07) with their respective ap-

proximate LD,, values: 14:16:22:24 g I k g in
the mouse. Reactions of serine proteinases
(e.g., thrombin) will activate the coagulation
cascade, but nonionic contrast media may
bind to the proteins to inhibit the clotting
(710). X-ray analysis of crystals of iohexol and
serine proteinase (pancreatic porcine elastase)
reveals that three molecules of iohexol are as-
sociated with elastase, with one close to the
active site (subsite Sl), the second in the vicin-
ity of Ser214in subsites S21S3, and the third
located in a pocket at the surface of the pro-
tein. The association is a result of the affinity
of iohexol directed toward the hydrophobic re-
gions of the enzyme and supports the hypoth-
esis of the contrast medium's potent inhibi-
tion of thrombin. Another example of the
contrast medium-protein interaction is be-
tween iopamidol and fibrinogen or lysozyme
(711). Isothermal calorimetry revealed a weak
endothermal effect at high concentrations of
iopamidol for both proteins. This endothermal
effect does not appear to be attributable to a
direct protein-iopamidol association but sug-
gests the alterations of protein solvation after
protein unfolding in the presence of contrast
medium. This altered solvation is confirmed
by Raman spectroscopy on two amino acids in
the presence of iopamidol, which showed that
the spectrum of alanine is unchanged at any
iopamidol concentrations studied, whereas
the spectrum lines attributed to the thiol
group of cysteine are shifted in a manner con-
sistent with altered solvation. A recent report
on the binding chemistry of 2,3,5-triiodoben-
zoic acid (TIB) with the crystal form of human
serum albumin (HSA) revealed that the ligand
TIB is bound in a hydrophobic crevice in a
cavity in subdomain IIA of the HSA structure
(712).
6.5.9 Histamine Release. Contrast media
administered to laboratory animals by either
intravenous injection or infusion or local ap-
plication caused histamine-like reactions: en-
dothelial injury (713),changes in vascular per-
meability (714, 715), and perivascular edema
(716). These agents release histamine from
tissue mast cells by degranulation (717-720).
Leite et al. (721) reported that meglumine io-
dipamide and a mixture of sodium and meglu-
mine salts of acetrizoate were more effective
Radiopaques
than iodamide in histamine release in the rat,
and diatrizoate was ineffective. Others have
reported the release of histamine from rat
peritoneal mast cells by acetrizoate, diatrizo-
ate, and iothalamate (722, 723). Histamine
was found in the plasma of dogs after pulmo-
nary artery injection of meglumine iodipamide
(722) and in the perfusion of canine lung with
meglumine salts of acetrizoate, diatrizoate,
iothalamate, and iodipamide, or meglumine
chloride alone (527). The ability or inability of
diatrizoate to release histamine reported in
these experiments is probably caused by the
difference in injection rate. Meglumine salts of
contrast media, with the exception of diatrizo-
ate, release much greater quantities of hista-
mine than do the sodium salts. Histamine re-
lease is not caused by hyperosmolarity, given
that the injection of isosmotic sodium chloride
(721) or dextrose (724) solutions produces
negligible effects. Administration of diphen-
hydramine hydrochloride and burimamide
i . . , N-methyl-N'-[4-[4(5) imidazolyllbutyll-
thiourea), which are H, and H, receptor an-
tagonists, respectively (725),abolishes the his-
tamine release properties of the contrast
agents in vivo (721, 726).
In humans the allergic reactions from con-
trast media resemble those elicited by hista-
mine injection, and the unpleasant side effects
are rarer after fast injection than after siow
injection of the same dose of contrast medium
(489). Seidel et al. (724) showed that usual
doses of chemically different contrast media
were able to affect the plasma histamine levels
in venous blood of healthy persons. Histamine
release ceases when the dose rate is reduced to
0.17 g kg-' min-l. This may explain the ad-
vantage of administering urographic agents
by infusion to avoid reactions.
Patients with a history of allergy react
more frequently to injection of contrast media
than those with no history of hypersensitivity
(455, 456). Blood of patients with radiocon-
trast idiosyncrasy and atopic individuals re-
lease higher levels of histamine than that of
normal individuals. Siegle et al. (727) incu-
bated 1251-diatrizoatewith blood from atopics
and nonatopics and found significant reten-
tion of radioactivity by the leukocytes and not
by the erythrocytes. Histamine is released
from human leukocytes by contrast media.
6 Organic Iodine Compounds
The low osmolality contrast media, such as
iohexol, iopamidol, metrizamide, and ioxa-
glate, release less histamine on the basis of
iodine concentration than the ionic contrast
media, such as diatrizoate, sodium, and meg-
lumine iothalamate. The meglumine salt re-
leases more histamine than the sodium salt,
and ioxaglate releases more histamine than
iohexol, iopamidol, and metrizamide (728).
Because the exact mechanism relating hista-
mine release to adverse effects of contrast me-
dia is unclear, cholinergic mechanisms have
been proposed to play an important role also.
These mechanisms are activated by the inhi-
bition of cholinesterase. Again, iohexol and io-
pamidol cause less enzyme inhibition than
ionic ioxaglate, iothalamate, and ioglycamide
(729). An in uitro assay based on histamine
release to pretest patients at high risk before
infusion with contrast medium has been pro-
posed (730), but the rise of plasma histamine
levels cannot be correlated with allergic-like
effects in patients in a conclusive manner
(731). Studies by Pinet et al. (732)showed that
the values of plasma histamine and blood his-
tamine in patients receiving ioxaglate, io-
hexol, iopamidol, and ioxithalamate for intra-
venous pyelography were within the limits of
statistical fluctuation. Reactions to contrast
media are in some cases precipitous, resem-
bling anaphylactoid reactions of immunologic
nature, which led Brasch (551,552) to suggest
an antibody-type reaction to contrast media.
Krause et al. (733), from the results of
Brasch's study, suggested that iotrolan is
probably not immunogenic.
6.5.1 0 Pharmacokinetics. The quality of
roentgenograms in cholangio-cholecystogra-
phy and urography is determined by the local-
ization of the contrast media applied. In oral
cholecystography, the radiographic quality is
determined by the absorption and excretion of
contrast medium, which is controlled by many
factors (734, 735). In intravenous cholan-
giography and urography, the excretion of
contrast medium alone determines the
opacification of the organ under examina-
tion (736-739).
Absorption of oral cholecystographic agents
occurs by passive diffusion of the nonionized
compound across the lipoidal barrier of intes-
tinal mucosa (734) and is determined by the
rate of solubilization of contrast medium, af-
fected by pH, the presence of bile salts, mixing,
the effective surface area, and the physical
state of the compound (735). The rate of ab-
sorption of iopanoic acid and iosumetic acid is
increased if the contrast agent is administered
concomitantly with sodium bicarbonate (300,
740, 741). Bile salts or a fatty meal have been
recommended with the administration of io-
panoic acid, to ensure its solubilization and
absorption in the duodenum and to improve
gall bladder visualization (735, 742, 743). The
fatty meal releases cholecystokinin, which
promotes the enterohepatic circulation of bile
salts, thus enhancing both intestinal absorp-
tion and hepatic excretion (734). The ability of
bile salts to increase the excretion of iolsanoic
acid is attributed to an allosteric interaction
between the bile salts and a carrier for the
contrast medium in the canalicular membrane
(744, 745).
Because of the often incomplete absorption
of oral cholecystographic agents, detailed an-
alytical approaches to pharmacokinetics stud-
ies have not been available. Goldberg et al.
(735) studied the biopharmaceutical factors
influencing the intestinal absorption of io-
panoic acid and attributed the frequent failure
of the first dose of the agent to afford diagnos-
tic visualization of gall bladder to impaired in-
testinal absorption. Uncertainty in absorption
is avoided when intravenous agents are used.
Water-soluble dimers such as iodipamide,
ioglycamide, iodoxamate, and iotroxic acid are
intravenous cholangiographic agents and can
be injected to achieve a desired plasma concen-
tration. After injection, the pharmacokinetics
of these contrast media including dilution and
mixing with body fluid, uptake by the liver,
and excretion into the bile and urine can be
fitted to multicompartmental kinetic models.
A logical kinetic model for the transfer of a
substance through a secreting cell may consist
of three phases: uptake (passage from the
plasma into the cell); passage through the cell
(with or without accumulation); and passage
from the cell to the tubular lumen (excretion)
(746,747). Corman et al. (748) have proposed
several pharmacokinetic models for ioglyca-
mate. Although a simple, two-compartment
model can account for approximately 90% of

lnjected dose
1 in direct communication with the plasma com-
Urine &le
lnjected dose
1 (U), B/U excretion ratio, and excretory deficit
II
1
Urine lnjected dose 1
Bile
I tion and the concentration of iodine in the bile
Urine ~ile
Figure 10.1. Pharmacokinetic models for the ex-
cretion of iodipamide, iodoxamic acid, and ioglycam-
ide in the dog (747-752).
the administered drug, the experimental data
-
are better fitted to three-com~artment mod-
els. The introduction of an extravascular
space with which the plasma contrast medium
can rapidly exchange would avoid the diffi-
culty of requiring a considerably larger initial
volume of dilution for the contrast medium
than the plasma volume in a two-compart-
ment model (748) and is based on the observa-
tion that the distribution volume of iodipam-
ide in the dog is similar to the extravascular
fluid volume in size (749). Pharmacokinetic
models involving three compartments have
been proposed for ioglycamic acid by Corman
et al. (747), for iodipamide by Shames et al.
(748), and for iodoxamic acid by Strata et al.
(750). Segre and Rosati (751) have proposed
nonlinear kinetic models for the disposition of
iodipamide, ioglycamide acid, and iodoxamic
acid in dogs. These models are shown in Fig-
ure 10.1. In the nonlinear kinetic model, the
Radiopaques
contrast medium is excreted into the bile
through the liver compartment 3, which is not
partment 1. For ioglycamic acid at doses above
175.5 ~mol/kg,an alternative model has been
proposed in which the passage of the contrast
medium from blood to the liver is irreversible.
The relationship between infusion rate or
plasma concentration on the one hand and bil-
iary concentration (B), urinary concentration
(i.e., the amount of contrast medium retained
in the body) on the other have been studied in
the case of iodoxamate and iodipamide by
Berk et al. (752) in unanesthetized dogs. Bili-
ary excretion depended on bile flow and eryth-
ritol clearance (which serves as an index of
canalicular bile flow). The rate of biliary excre-
increased according to first-order kinetics at
low plasma concentration of the contrast
agents and leveled off when the plasma con-
centration reached 0.4 mol/mL. A reasonable
fit of these data to equations for hyperbolas
can be made, indicating saturation kinetics,
which is characteristic of an active transport
process presumably occurring at the biliary
canaliculus. The rate of urinary excretion of
these contrast agents varied linearly with the
plasma concentration, indicating a passive
transport process. The B/U excretion ratio de-
creased sharply with increasing plasma con-
centration (i.e., above 0.4 mol/mL). At an infu-
sion rate of approximately 1 mmolkg min, a
biliary concentration of about 25 and 28
mmol/mL has been achieved for iodipamide
and iodoxamate, respectively, which exceeds
the concentration (approximately 13-16 mmol/
mL) required for visualization of the biliary
tree. From these data, the authors suggested
that iodoxamate is a better agent than iodip-
amide in patients with impaired biliary excre-
tion, high basal bile flow, or when a small dose
of the contrast medium is infused. Lin et al.
(753) have investigated the saturation kinet-
ics of iodipamide. Moss et al. (754) found that
iopanoate and iodoxamate, when co-adminis-
tered intravenously in rhesus monkeys, com-
pete for the binding sites on serum proteins as
well as on intrahepatic tissue and that biliary
excretion is directly related to the unbound
contrast medium in the blood. Staubus (755)
6 Organic Iodine Compounds
simulated the nonvisualization of gall bladder
in the patients by studying the effects of infu-
sion on the pharmacokinetics of iopanoate in
the dogs and attributed the nonvisualization
to poor intestinal absorption or to poor he-
patichiliary function.
Pharmacokinetic studies of urographic
agents such as water-soluble derivatives of
2,4,64riiodobenzoic acid and 2,6-diiodopyri-
done showed that the renal excretion of these
agents proceeds through glomerular filtra-
tion, active excretion in the proximal tubules,
and passive reabsorption in the distal tubules
of the nephron (756). Iodopyracet and ace-
trizoate are largely excreted by the proximal
tubules in the dog, whereas iodamide is
only moderately excreted; and diatrizoate,
iothalamate, and metrizoate are not excreted
at all by the tubules but are eliminated by glo-
merular filtration. Studies with aglomemlar
fish or ureterally obstructed dogs confirmed
that there is no tubular excretion or reabsorp-
tion of sodium iothalamate (757).
The plasma concentration curve obtained
after a single injection of sodium diatrizoate
reflects the initial mixing in the vascular com-
partment, equilibration in the fluid space, and
concomitant excretion through the kidneys.
The initial volume of mixing is of a size com-
parable to the measured extracellular space.
Based on the excretion data of l3'I-labeled so-
dium and meglumine diatrizoate, a three-com-
partment kinetic model, analogous to that
proposed for iodipamide excluding the biliary
exit has been developed using analog com-
puter simulation. The model is composed of a
blood compartment, a tissue compartment,
and a "deep" tissue compartment (738). The
deep tissue compartment corresponds to the
extracellular fluid space and is characterized
by a very small entry constant and a large en-
try-to-elimination constants ratio, usually ex-
ceeding 10. This ratio was found to be 9.4 ?
4.5, with a half-time of elimination of 11.7 ?
1.15 h for sodium diatrizoate compared to 17
t 4.5 and 14.5 ? 1.5 h for the meglumine salt.
These values show that the pharmacokinetic
model for the diatrizoic acid remains the same,
unaffected by its salt form.
The plasma concentration C, of the non-
ionic monomer iohexol, as shown in two stud-
ies in human volunteers, was best represented
by a three-compartment model, C, = AePmt+
BepPt + CeCYt,where A, B, and C are the
feathered intercepts of the plasma concentra-
tion curve; a, P, and y are the rate constants;
and t is the time (758, 759). In one study the
mean apparent first-order terminal (y-phase)
elimination half-life for iohexol was estimated
to be about 12.6 h; the half-life of initial distri-
bution (a-phase, tll,,) about 22 min, and the
half-life of disposition and elimination (P-
phase, tllZp)about 2.1 h. The mean + SD ap-
parent volume of distribution (V,) was calcu-
lated to be 165 ? 30.7 mL/kg body weight. The
magnitude of the value suggests that iohexol
was distributed in a volume comparable to
that of extracellular water. The renal clear-
ance (CLR) was 120 ? 18.6 mllmin and the
total body clearance (CLT) was 131 + 18.6
mL/min. In a separate study, the t,,,, value of
iohexol was estimated to be an average of 21.8
rnin and the tllZpvalue an average of 121 min;
the CLR was calculated to be 122 (118-128)
mL/min and the CLT, 120 (116-118) mLlmin.
Nonionic monomers, such as iopamidol (195,
760), ioversol(761,762), iopromide (7631, and
iopentol (764) have similar pharmacokinetics
profiles in healthy subjects. The mean plasma
concentration curve of iopamidol at three dose
levels in healthy volunteers was best fitted to
an open, linear, two-compartment model with
an average t,,, value of 28.2 min and an aver-
age tllZp of 2.14 h (699). Ioversol given to
healthy volunteers by intravenous injection at
two dose levels (50 and 150 mL) showed the
~ I I Z , and tlIzPvalues to be 15.5 min and 2.06 h
for 50-mL dose group and 18.9 rnin and 2.04 h
for 150 mL dose group, respectively (762). The
CLR and the CLT were 178 and 156 mL/min
for the 50-mL dose group and 190 and 184 mL1
min for the 150-mL dose group, respectively.
The pharmacokinetics of iopromide was fit-
ted in a two-compartment model in healthy
volunteers at low dose (15 g iodine150 mL) and
high dose (80 g iodine150 mL) levels and their
t112, and tll,p values were 1.95 and 2.6 h for the
low dose group and 1.90 and 2.5 h for the high
dose group, respectively (763). The CLR and
the CLT were 104 and 110 mllmin for the low
dose group and 100 and 103 mllmin for the
high dose group, respectively. The pharmaco-
kinetic parameters of iopentol were 23 min for
tll,, and 121 rnin for t,,,,. The apparent vol-

ume of distribution was 250 mL/kg of body
weight; the CLR was 104 mL/min, and the
CLT, 106 mL/min. The nonionic dimer iodixa-
no1 showed the same profile in pharmacoki-
netics as that of the nonionic monomers. The
serum concentration of iodixanol declined
biexponentially after intravenous administra-
tion of the dose. The t,,, and tIlzpvalues were
25.6 and 130.6 min, respectively. The average
apparent volume of distribution was 0.275
L/kg body weight. The renal clearance and the
total body clearance were 104 and 106 mL/
min, respectively.
Urinary excretion of water-soluble con-
trast agents is by glomerular filtration with-
out any tubular reabsorption (689). The CLR
for the contrast medium was compared with
the clearance of 51Cr-EDTA,injected alone or
co-injected with the contrast medium, given
that 51Cr-EDTAis known for its excretion by
glomerular filtration only (759, 765). The
elimination half-life tllzp usually expresses
urinary excretion in a comparable term for
water-soluble contrast agents. In addition to
the values given above, iotrolan has a tlDp of
106 min and diatrizoate, 108 min. According
to Olssen et al. (759),
[Tlhe apparent volume of distribution is an
important pharmacokinetic parameter relating
serum concentrations of a drug to the total
amount of drug in the body. The parameter usu-
ally has no direct physiologic meaning and does
not refer to a real volume. However, it is gener-
ally accepted that a volume of distribution of 15-
27% of body weight (0.15-0.27 Lkg) indicates
the distribution in the extracellular fluid only.
The volumes of distribution reported for
contrast media in the dogs are generally
higher than the reported human values, such
as 0.42 L/kg for ioxaglate, 0.37 L/kg for iocar-
mate, 0.64 L/kg for ioxithalamate, 0.39 L/kg
for mitrizamide, and 0.38 L/kg for diatrizoate.
Absorption and excretion of metrizamide
and diatrizoate after suboccipital injection in
the cat followed first-order kinetics. Golman
and Dahl(766) determined the rate constants
for absorption and excretion and observed
that 99% of the absorbed dose disappeared
from cerebrospinal fluid during the 3 h after
suboccipital injection. The difference in the to-
Radiopaques
tal amount of metrizamide excreted during
48 h after intravenous and suboccipital injec-
tion in the rabbit was negligible, indicating a
rapid removal of the agent from cerebrospinal
fluid to blood (767).
6.5.1 1 Excretion. Contrast media are ex-
creted in both the bile and urine; the route
that predominates is determined by their
chemical structure and route of administra-
tion. In general, contrast molecules with an
unsubstituted position in the benzene nucleus
will be sufficiently lipophilic to bind to the pro-
teins and will be absorbed in peroral adminis-
tration and excreted in the bile and urine. On
the other hand, if the benzene nucleus is fully
substituted, the contrast molecules will be
highly hydrophilic, will not bind to the pro-
teins, and will be rapidly excreted in the urine.
The nonionic contrast media are highly solu-
ble in water and will not be adequately ab-
sorbedper 0 s . Hepatic and urinary excretions
of ionic contrast agents and their rates of ex-
cretion have been reviewed by Catell (688),
Knoefel and Carrasquer (7681, McChesney
(769), and Sperber and Sperber (749).
Oral contrast agents bind serum protein at
physiological pH (417, 688). Protein binding
prevents glomerular filtration but does not
impair hepatic excretion. Transport of these
agents from the blood to the liver is an active
saturable process (770) and involves the Y and
Z proteins found in the liver as hepatic accep-
tors (694, 695). These acceptors show affinity
for cholegraphic agents, such as iopanoic acid
and iodipamide, but exhibit little or no affinity
for urographic agents such as iothalamate.
The liver transport capacity of contrast me-
dium, expressed as maximum transport rate
or velocity Tm, is defined as the amount of
compound going into the bile per unit time,
which cannot be influenced by additional in-
creases of dose (190). Rosati et al. (190) re-
ported the Tmvalues for ioglycamic acid, iodi-
pamide, and iodoxamic acid after rapid
intravenous injection in the dog to be 0.768 ?
0.101, 0.530 ? 0.072, and 1.226 ? 0.109 pmol
kg-' min-l, respectively. These values re-
main essentially the same for slow infusion
(738, 771). Iopanoate has a T , value varying
from 0.16 to 0.74 mol kgp1 min-l, depending
on the presence of bile salts (772). In bile salt-
6 Organic Iodine Compounds
depleted dog, less contrast agent is excreted.
Julian et al. (773) reported the maximum
transport rates of iodipamide and iodoxamic
acid in humans.
Contrast agents with strong affinity for se-
rum protein show a threshold plasma value
below which little or no excretion through the
liver or the kidney occurs (771), and in addi-
tion, a species difference exists in the excre-
tion of cholegraphic agents (774). The thresh-
old values of ioglycamic acid and iodipamide in
the dog were shown to be 0.16 and 0.6 pmoll
mL, respectively. Iodoxamic acid, iodipamide,
and ioglycamic acid were excreted in the rab-
bit at higher concentrations to the extent of
about 77, 38, and 42% of the dose in the bile
and about 17, 38, and 35% of the dose in the
urine, respectively (190). Iocetamic acid is ex-
creted in the rat predominantly in the intes-
tine but in human subjects, 41 and 62% of the
administered dose appear in the urine in the
first 24 and 48 h, respectively (774). Kiyono et
al. (775) studied the distribution and excretion
of iodipamide in mice and man, using 1311-la-
beled material. Earlier, Fischer (776) derived
from biliary and urinary excretion studies an
optimal dose of 205 pmolikg for iodipamide.
Biliary excretion of contrast medium is af-
fected by the bile flow (688, 734, 745, 777,
778). Bile is isosmotic with plasma and is pro-
duced from the transport of water from the
liver cell into the bile canaliculi (canalicular
bile flow) and from the excretion and reab-
sorption of water and electrolytes in the bile
ductules (ductular bile flow). Bile flow is in-
creased by taurocholate and dehydrocholate;
their presence in the canaliculi creates an os-
motic gradient that produces the flow of water
and solute. There is a positive correlation be-
tween the canalicular bile flow stimulated by
taurocholate and the amount of iopanoic acid
excreted by the liver. Feeding the patient a
fatty meal or taurocholate at the time that io-
panoic acid is administered can improve the
quality of cholecystograms (734).
In addition to bile salts, contrast media
(779) and other agents (752, 777) may also in-
crease bile flow and cause choleresis. Iodipam-
ide produces 0.025 mL bilelpmol excreted
compared with 0.009 mL bile/pmol for ipo-
date, but both agents are able to achieve sim-
ilar maximum bile iodine concentration. The
reason for this phenomenon is that ipodate is
excreted as glucuronide conjugate, incorpo-
rated in the bile salt micelles, which exert a
small choleretic effect, whereas iodipamide is
excreted unchanged (780).
Drugs such as phenobarbital and cincho-
phen alter the bile flow and the biliary excre-
tion of cholegraphic agents. Pretreatment
with phenobarbital decreases iodipamide bile
excretion and increases iopanoate bile excre-
tion (752). Cinchophen increases both biliary
and urinary excretion of iophenoxic acid but
has no effect on iopanoic acid (777). It has been
reported recently that a high level of prosta-
glandin-like substance in the mucosa and
muscle wall of gall bladder caused nonvisual-
ization of the gall bladder in cholecystography
(781).
Urinary excretion is the principal means of
elimination for diatrizoate, iothalamate, met-
rizoate, metrizamide, and other urographic
agents (688, 767, 782, 783). Diatrizoate,
iothalamate, and metrizamide were excreted
in the urine of the rabbit to the extent of more
than 80% 24 h after injection (768, 783-785).
The total recovery including the amount ex-
creted in feces 96 h after injection was about
91.5% of the dose for metrizamide, 93.8% for
diatrizoate, and 86.0% for iothalamate (782).
The nonionic contrast media are highly hy-
drophilic. When given orally, iohexol is ab-
sorbed from normal bowel (785). In dogs, ap-
proximately 2% of the oral dose is absorbed
and excreted in the urine within 6 h, and about
4% of the dose is absorbed and excreted in cats.
Using lZ5I-labelediohexol, Miitzel and Speck
(267) found intestinal absorption to be about
2% of the intravenous dose given at 60 mg
I/mL body weight to male rats. The urinary
excretion from the rat was 91.5 f 3.6% of the
administered dose of lZ5I-labelediohexol, and
the fecal excretion 6.8 + 2.7% of the dose 24 h
after intravenous injection. In the dog the
urine contained 81 ? 9% of the administered
dose within 3 h after injection. Total recovery
from urinary excretion in the dog during the 7
days after injection was 98 f 4% of the dose
and total recovery from fecal excretion was
0.95 ? 0.45%. Healthy volunteers excreted
84% of the dose in urine within 4 h and 100%
of the dose within 24 h after intravenous injec-
tion at the rate of 25 mLlmin of iohexol at a
564
solution concentration of 350 mg IImL at one
of the dose levels (i.e., 100, 200, 375, and 500
mg I/kg body weight) (733). Another group of
healthy volunteers were administered iohexol
at higher dose levels (i.e., 500, 750, 1000, and
1500 mg I/kg body weight) and excreted 92.3 +
4.44% of the dose in urine 96 h after injection,
with 89.9 + 7.0% of the dose appearing in
urine within the first 12 h (758).
The biodistribution of 1251-iobitridolwas
studied in rats using whole-body autoradiog-
raphy (786). Radioactivity was found mainly
in the kidneys, skin, and thyroid. The accumu-
lation of radioactivity in the thyroid was at-
tributed to free 1251-iodideformed in radiola-
beling. Iobitridol, a marker of extracellular
fluid, was excreted totally in the urine by glo-
merular filration within 48 h after intrave-
nous injection. The CNS showed no uptake.
Bourrinet et al. (787) studied the excretion of
iobitridol or iohexol in goat milk after an in-
jected dose of 300 mg I/kg and found 0.7% of
the administered dose in the milk for iobitri-
dol, compared with 1.6% for iohexol. In the
gestating rabbit concentrations of both con-
trast agents fell rapidly below the detection
limit (0.2 mg IImL) 180 min after injection.
Concentrations in fetal plasma and amniotic
fluid were not measurable up to 24 h for both
contrast agents.
The excretion of nonionic iopamidol was
studied in healthy subjects at several dose lev-
els by use of two different solution concentra-
tions of 200 and 300 mg I/mL, which were ad-
ministered intravenously at a rate of 20 and 39
mllmin, respectively (760). Urinary elimina-
tion of iopamidol was rapid at all dose levels,
with more than half of the dose excreted by the
kidneys in the first 2 h after injection. Total
urinary recovery within 72-96 h after injec-
tion was more than 90%, and the fecal recov-
ery for the same period was about 1% of the
administered dose. 14C-Labelediopamidol was
also included in this study for quantitation
and the search for metabolic products of io-
pamidol, of which none was found. In dogs the
urinary and fecal excretion of iopamidol at
72 h were 94.8 ? 1.2 and 0.63 2 0.32%,respec-
tively, of the injected dose at the dose level of
50 mg I/kg body weight. At a higher dose level
of 200 mg I/kg, these excretions were 93.4 +
1.5 and 3.17 + 1.39%,respectively (195). The
Radiopaques
excretion of another nonionic contrast me-
dium ioversol, labeled with iodine-125, was
studied in rats. The elimination of radioactiv-
ity from the blood was extremely rapid. Uri-
nary excretion was 95.33% and fecal excretion
was 3.8% of the administered dose within 48 h
after intravenously injection. Urinary excre-
tion of ioversol in healthy volunteers was com-
plete at all dose levels within 24 h after intra-
venous administration (762).The excretion of
a dimeric nonionic contrast agent iodixanol
was investigated in healthy volunteers at four
dose levels (i.e., 0.3, 0.6, 0.9, 1.2 g I/kg body
weight). Within 24 h after injection, 97% of the
dose was excreted unmetabolized in the urine
and 1.2% in the feces (765).
These water-soluble contrast agents are ex-
creted as rapidly as glomerular filtrate can be
formed (688). Their elimination may - be im-
paired by primary and secondary glomerular
dysfunction or reduced renal blood flow that
diminishes the filtration rate (788). After
clearing the blood, the contrast medium in its
passage down the nephron can affect the salt
and water reabsorption in the proximal tubule
and water reabsorption in the distal tubule.
The osmotically active contrast medium can
produce salt and water diuresis, and its pres-
ence in the nephron may still cause renal tox-
icity (789). In experimental animals (790)and
in humans (688), the sodium salt of diatri'zoic
acid produces less diuresis than that of the
meglumine salt. The renal extraction ofp-ami-
nohippurate is reduced by the contrast anion
and the meglumine cation (791). Iodamide, an
intravenous pyelographic agent, is rapidly and
almost completely excreted in the urine; in pa-
tients with moderate to severe renal impair- -
ment, it is still excreted in the urine but at a
much slower rate (792).
The excretion of contrast agent by the liver
or the kidney is not exclusive; when either of
these organs is in a dysfunctional or diseased
state, heterotopic excretion by the other organ
will occur to compensate for the impediment
(688, 781, 783). In patients with renal failure
or advanced renal disease, contrast media may
still be used to produce nephrograms (689); if
the tubular obstruction prevents the passage
of urine along the nephron, back-diffusion of
the contrast medium into the interstitial vol-
ume will occur (791,793,794). During the pe-
6 Organic Iodine Compounds
riods of ureteric stasis, metrizarnide is ex-
creted faster than diatrizoate, thus producing
a higher urinary iodine concentration (685,
795). Extrarenal clearance of water-soluble
contrast media is very low and can be practi-
cally neglected (796).
When suboccipitally injected, metrizamide
was recovered in the urine and feces of rabbit,
rat, and cat to the extent of more than 90% of
the dose (787), similar to the recovery of
iothalarnate (797) and iocarmate (798)in dogs.
In peroral administration, metrizamide
and diatrizoate are not well absorbed from the
GI tract; the urinary recovery of both agents
in the rabbit was below 5% of the administered
dose, 24 h post-administration (767). The non-
ionic contrast media are highly hydrophilic
and cannot cross the lipophilic cell mem-
branes in the intestine to be absorbed as an
oral dose. Iophendylate, when given to rats,
guinea pigs, or rabbits, was eliminated from
the body in 5 h (799). It was transported along
the lymph system.
6.5.1 2 Biotransformation. Contrast media
may be excreted either metabolized or un-
changed, depending on their molecular prop-
erties. McChesney (800) has reviewed the sub-
ject of biotranformation of contrast media.
The fully substituted 2,4,6-triiodobenzoic
acids used for angiography and urography
have high water solubility and are stable
chemically. Because of their low pK, values,
they are highly ionized at physiological pH.
These contrast agents are poorly absorbed
from the gastrointestinal tract and rapidly ex-
creted unchanged within 5-6 h after intrave-
nous injection (776). Their metabolic inert-
ness is attributed to their inability to cross cell
membranes and to penetrate liver microsomes
to undergo biotransformation (800). Their
rapid excretion is caused by the absence of tu-
bular reabsorption, thus allowing as rapid an
excretion as the glomerular filtrate can be
formed. Diatrizoate (3,5-diactamido-2,4,6-tri-
iodobenzoate) can be partially deacetylated to
3-amino-5-acetamido-2,4,6-triiodobenzoate
(I) and 3,5-diamino-2,4,6-triiodobenzoate (11)
by liver microsomes of the rabbit and guinea
pig (801). The deacetylated products are rap-
idly excreted. In the urine of patients only the
presence of (I) is confirmed.
In contrast to the 2,4,6-triiodobenzoic ac-
ids, substituted alkanoic and alkoxyalkanoic
acids containing a 2,4,6-triiodophenyl or a
2,4,6-triiodophenoxygroup with open position
5, such as iopanoic acid, tyropanoate, iogly-
camic acid, and iopronic acid, have both li-
pophilic and hydrophilic properties. These
contrast acids have an acidity constant about
two units higher than that of the water-
soluble, fully substituted 2,4,6-triiodobenzoic
acids (263) and are not completely ionized at
the physiological pH. In the nonionized form,
-
the contrast molecules can be absorbed and
transported across cell membranes after oral
administration.
The oral cholecystographic agents are in
general more toxic than angiographic and uro-
graphic agents. Detoxification of the former
may involve glucuronidation, N-acetylation,
0-acetylation, esterification, deiodination,
and hydrolysis. These metabolites appear in
the bile and urine. The metabolites of many
cholegraphic agents have been characterized
to some degree but are not chemically
identified.
Conjugation with glucuronic acid occurs
with most oral cholecystographic agents be-
fore excretion. Bunamiodyl(802,803), iodoal-
phionate (804, 805), iolidonic acid (8061, io-
phenoxic acid (807-809), and tyropanoate ,
(802, 803) are excreted as glucuronides. Mc-
Chesney and Banks (803) showed that 90% of
the contrast agents in the urine appeared as
glucuronides. Because glucuronides are usu-
ally poorly reabsorbed from the intestine, gluc-
-
uronidation can thus circumvent extensive
enterohepatic recirculation of the contrast
medium.
The fate of iophenoxic acid (809) and the
efficacy of iopanoate (800, 802) are uniquely
linked to glucuronidation. Iophenoxic acid is
retained in the body over a period of many
years after administration and is slowly ex-
creted. It forms acyl and ethereal monoglucu-
ronides and the diglucuronide. These glucuro-
nides have the uncommon property of being
freely lipid soluble and can undergo hepatic,
intestinal, and renal tubular reabsorption.
Wade et al. (809) pointed out that the rate of
formation of glucuronide conjugates may be
an important factor in the persistence of io-
phenoxic acid in the body. Iopanoate glucuro-
566
nide, when administered orally, gave no visu-
alization of gall bladder but when injected
intravenously yielded an excellent cholecysto-
gram 1 h after administration; however, an
equivalent dose of iopanoate when injected re-
quired a delay of 8 -24 h for an image of diag-
nostic quality to become apparent, indicating
that the iopanoate was converted to glucuro-
nide before being excreted in the bile (800,
802).
Bornschein et al. (297, 810) reported the
appearance of the unchanged iomeglamic acid,
the methyl ester, the deiodinated derivative,
the N-desmethyl derivative, the methyl ester
of the latter derivative, the N-acetylated deriv-
ative, and the deiodinated methyl ester of
iomeglamic acid in human urine 72 h after a
3 g dose. Lindner et al. (293) showed evidence
of biotransformation of iobenzamate in the
mouse, rat, and human, which involved conju-
gation with glucuronic acid, hydroxylation of
the unsubstituted phenyl ring, or hydrolysis of
the amide linkage, for example, but isolation
and identification of the metabolic products
were not carried out. Harwart et al. (286)
studied the metabolic fate of i ~ o d a t eand
found that there was unchanged ipodate in the
bile, and the major product exereted in urine
was soluble in butanol. One of the metabolites
. . -
was identified as 3-amino-2,4,6-triiodohydro-
cinnamic acid, a reduction product of ipodate.
The hexaiodinated dimeric intravenous
cholangio-cholecystographic agents, such as
iodipamide, ioglycamide, iodoxamate, and
iotroxic acid, are excreted mainly unchanged
(788, 776, 811-814). Strickler et al. (815) re-
ported an unidentified metabolite of iodipam-
ide, probably formed from hydrolysis of the
amide linkage. Miitzel et al. (816), by use of
l3'I-labeled tracers, found that ioglycamide,
iodoxamate, and iotroxic acid each yielded two
urinary metabolites, one of which was more
polar and the other less polar than the parent
molecule. It was shown that neither of these
two metabolites was a deiodinated product,
and that the bile and plasma contained no me-
tabolites. Pitre and Felder (817) reported
three metabolites of iodoxamic acid in human
urine, one of which was identified as 3-[3(a-
hydroxyethoxy)-propionamidol-2,4,6-triiodo-
benzoic acid, a product formed from symmet-
rical scission of the dimer.
Radiopaques
Deiodination is not an important reaction
in the biotransformation of contrast media, in
spite of the presence of deiodase in the mam-
malian liver (818). The rat and pig liver, how-
ever, can convert 2,3,5-triiodobenzoic acid
into 2,5- and 3,5-diiodobenzoic acids (819-
821). Iodoalphionate (822, 823), iothalamate
(824), iodipamide, and ioglycamide (812) were
reported to undergo deiodination to a small
degree, never exceeding 1% of the dose. Be-
cause most organic iodine compounds are sen-
sitive to light, the observed deiodination may
be a result of both enzymatic reaction (821)
and photolytic degradation (825). Wong (826)
studied the kinetics of deiodination of the wa-
ter-soluble radiopaques diatrizoate and iopam-
idol, and found that deiodination proceeds by a
Cu(I1)-catalyzed S Nmechanism
~ and that the
copper complexes specifically with the ortho-
carboxylic acid group. Nonionic iopamidol
without the o-carboxylate group undergoes
deiodination by S Nmechanism
~ involving hy-
droxyl ion, and the rate is extremely slow. Ki-
netic data show that the S Nmechanism
~ may
still be participating in deiodination. Deiodin-
ation in both contrast agents is prevented by
the presence of sodium edetate (0.04%).
Deiodination of contrast agents may affect
the thyroid 1311uptake. Iodoalphionate (827,
828), iodipamide (827, 829), and iodopyraqet
(830) affect the thyroid uptake by a slow re-
lease of the iodide. In thyroid patients, the thy-
roid uptake returns to normal 4 days after the
administration of acetrizoate, 58 days after io-
panoic acid (8311,and many years after iophen-
oxate (832). Iophenoxate binds avidly to
plasma protein and is transported across the
placental barrier as long as 5 years after ad-
ministration. The cholecystographic agents
may have a direct effect on the thyroid. Iodide-
induced hyperthyroidism may result from ipo-
date administered for cholecystography (833)
or from iophendylate injected for myelography
(834). Intravenous administration of ioxita-
lamic acid to euthyroid patients increased
their protein-bound iodine (PBI) and hor-
monal iodine levels, which returned to normal
between 2 and 8 days (835).
6.6 Uses
Roentgenographic procedures requiring the
use of radiopaques are listed in Table 10.10.
6 Organic Iodine Compounds

Table 10.10 Uses of Contrast Agents:

- aging. The strengths and problems of the cur-
Radiographic Procedures for Visualizing rently available imaging techniques in their
Organs After Administration book are compared, to which the reader may
of Contrast Agents
refer. Illustrative examples of the use of radio-
Procedure Organs Visualized contrast media for some roentgenographic
Blood vessels procedures, current or dated, are given below.
Arteries
Aortography Aortas
Arthrography Joints 6.6.1 Computer-Assisted Tomography KT).
Bronchography Lungs Unlike the conventional roentgenographic
Cholangiography Gall bladder and bile system in which the transmitted X-ray image
ducts is directly projected onto a fluorescent screen
Cholecystography Gall bladder or a photographic plate, in the CT system an
Esophagography Esophagus object is scanned with a narrow beam of X-
Hepatography Liver
rays from a multitude of angles, and the ab-
Hepatolienography Liver and spleen
Hysterosalpingography Uterus and oviducts sorption
. values of the material contained
Lymphography Lymph nodes and vessels within the object are computed from the X-ray
Lymphadenography Lymph nodes transmission readings and presented as a se-
Lymphangiography Lymph vessels ries of -pictures of slices of the object
- (837).The
Myelography Spinal cord, image reconstruction requires fast computing.
subarachnoid space The speed of the CT scanners has improved
Pelviagraphy Pelvis
Phlebography Veins
from the first-generation 4.5-min head-only
Pyelography Kidney and ureter scanner to the fourth-generation 2-s head or
Splenography Spleen body scanner (838, 839). The ultrafast scan-
Splenohepatography Liver and spleen ners that may be categorized as the fifth-gen-
Urography Urinary tract eration X-ray CT scanners are necessary for
Venography Veins the imaging of moving organs, such as the
Ventriculography Ventricles of the brain heart.
The first CT system was shown to be ap-
proximately 100 times more sensitive than the
.
Before the development of other medical
imaging systems, the roentgenographic proce- conventional X-ray system (837). Contrast en-
dures listed in Table 10.10 were performed hancement in CT is measured in terms of a
with radiocontrast agents. MRI with super- density unit. In the original article, water was
paramagnetic contrast agents, ultrasonogra- arbitrarily set at a value of 0 unit; air, 500
phy with echogenic contrast agents, and nu- unit; fat, 50 unit, and so forth. On this scale, a
clear medicine with radiopharmaceuticals, number of tissues in the body fell within 20
labeled with llC, 18F, 99mT~, and other short- units above water or "4% window" in scan-
lived radio-nuclides for example, are addi- ning. This unit was later named the
tional modalities for organ imaging and organ Hounsfield Unit (HU) in honor of the author
function studies. These systems have their (840). Using HUs, air has a value of -1000,
own merits, and their use led to the replace- water a value of 0, and dense bone a value of
ment of some of the above roentgenographic +1000. The white tone on the picture repre-
procedures. Progress in medical instrumenta- sents dense material with enhanced contrast.
tion has brought CT, helical CT, and the mul- Materials such as tumor will be less enhanced
tidetector-CT scanner into practice; these fast by the contrast medium because of limited
instruments greatly facilitate the use of radio- blood supply and will appear hypodense, gray,
contrast agents. The textbook of contrast me- or less white. Organs or tissues not contrast
dia by Dawson, Cosgrove, and Grainger (836), enhanced will appear black on the picture. A
published in 1999, contains up-to-date discus- similar unit, known as the CT number, for
sions of X-ray contrast agents, MR contrast measuring the image contrast or conspicuity,
agents, and ultrasound agents for medical im- discussed by Dawson (8361, is based on a com-
568
parison of the attenuation coefficient ( p ) of
the region of interest (ROI) to that of water
and is defined as:
~ R O Z- h a t e r
CT Number = transporting the patient through the gantry in
The increase in density in HUs or CT num-
bers is linearly dependent on the iodine con-
centration (mg IImL) of the injected contrast
medium. CT has two advantages over projec-
tion radiography. One is the significant in-
crease in contrast resolution and sensitivity,
which reduces the contrast detection thresh-
old, thereby allowing a lower level of con-
centration of contrast media t o be ad-
ministered; the other is the cross-sectional
imaging, which permits successive visualiza-
tion of structures without the superposition
of other tissues to affect the image quality
(632).
Computed tomography (CT) can detect
small differences in X-ray absorption at ap-
proximately one-fifth, perhaps as much as
one-eighth, of the concentration of contrast
media, if one uses lower kilovolt (kVp) tech-
niques, compared with that of conventional
radiography (841).Contrast media for CT may
be administered as bolus, or bolus followed by
infusion (biphasic), or infusion alone (842,
843). The pharmacokinetics is such that
within 1 min after bolus injection the contrast
enhancement reaches maximum in the ex-
travascular space, followed by a washout.
Computer-assisted tomography has been ap-
plied to angiography, arteriography, cardioan-
giography, phlebography, and numerous
other X-ray procedures. The sensitivity of CT
to minimal density differences between tis-
sues allows a variety of contrast agents, such
as xenon, air, saline, particulates, microemul-
sions, liposomes, and iodinated oils, to be ex-
plored for contrast enhancement. These in
combination with dynamic CT, in which con-
ventional water-soluble nonionic iodinated
contrast media are rapidly administered in in-
cremental amounts, are powerful means in ob-
taining diagnostic information (92,844, 845).
6.6.2 Spiral (or Helical) CT. Conventional
CT scans the body or organ volume by slices,
Radiopaques
whereas spiral CT scans the patient continu-
ously during a single breath hold, thus afford-
ing contiguity of slices and uninterrupted
scanning information. The spiral geometry in
CT scanning is achieved by continuously
synchrony with continuous data acquisition
over a multitude of 360" scans. Kalender et al.
(846, 847) used a standard continuous rotating
CT scanner that produces up to 12 consecutive
1-sscans and a modified table feed mechanism
with a stepper motor to allow continuous
transport of the patient at low, but accurately
controlled, speeds (0.1-11.0 mmls) during
scanning. The images are mathematically re-
constructed from the spiral geometry. Many
physical parameters, such as spatial resolu-
tion, image uniformity, linearity, and contrast
that determine the image quality of spiral CT,
are not affected, but noise is slightly reduced
and section sensitivity profiles are widened as
a result of transport during scanning. In spiral
CT the scan times for a complete volume are at
a minimum, and the scannings are contigu-
ous, independent of patient breathing and mo-
tion (847). Spiral CT provides speed, breath
hold protocols for less motion, fewer geo-
graphic misses, and easier timing of contrast
peak distribution (841). Hidajat et al. (848)
showed that spiral CT can lead to a reduced
radiation integral dose exposure to the patient
compared with that of conventional CT.
6.6.3 lonics versus Nonionics. Contrast
media are foreign substances, just like other
drugs, and thus when given in a large dose
within a short period, adverse reactions, ac-
companied by delayed symptoms perhaps, will
inevitably occur (849). McClennan (850) em-
phasized that mild and moderate reactions
should be given recognition and response be-
cause a minor side effect may be a prelude to a
more serious adverse reaction. Siegle (514)
compared the rates of idiosyncratic reactions
in patients receiving ionic and nonionic con-
trast agents and found that 4.4% of patients
receiving ionic agents experienced a reaction
as compared with 0.59% of patients receiving
iohexol. The percentage of patients requiring
treatment was 1.2% for ionic agents and 0.24%
for iohexol. Patients receiving ionic agents
plus steroids did not fare better. Cohen (851)
6 Organic Iodine Compounds
reviewed the toxicity of nonionic contrast
agents in children and found that moderately
severe reactions occur less frequently with
nonionic agents and that the incidence of ad-
verse reactions in children and in adults ap-
pears to be similar, contrary to observations
by others (553, 536, 630).
Bettmann (852) discussed the safety and
efficacy of iodinated contrast agents from the
viewpoints of use, regulation, and adverse
events and noted that an intra-arterial digital
subtraction angiography of the pelvis and
lower extremities performed with a contrast
agent, diluted to an iodine content of 100 mg
I/mL, yielded useful images for diagnosis. Di-
lution lowers the osmolality of contrast media
and improves tolerance. Dean et al. (853) stud-
ied the effect of dilution of diatrizoate (ionic
monomer), iopamidol (nonionic monomer),
ioxaglate (ionic dimer), and iodecol (nonionic
dimer) in a rat model at a dose of 500 mg I/kg
administered at two different concentrations
of 300 and 150 mg IImL. The authors observed
that the dilution of the contrast medium did
not lower iodine tissue concentration, iodine
distribution volumes, plasma volumes, or he-
matocrit, and should have no effect on CT con-
trast enhancement. Vergara and Sequel (630)
found that warming ionic contrast agents to
35°C before injection significantly decreased
the incidence of adverse reactions and that
ionic contrast agents without a sodium cation
caused significantly fewer reactions than with
a sodium cation. According to these authors a
nonionic, warmed contrast medium was the
best option for the outpatient CT examination
because no severe reactions resulted from its
use.
The cost differential between ionic and
nonionic contrast agents is substantial and is
a consideration for cost containment in health
maintenance. In 1994 in the United States
this cost differential is estimated to be about
10- to 20-fold, and it is far smaller in Europe
(852). The cost differential between nonionics
and ionics has been reduced to 5 or 6 to 1 in
2002 in the United States, according to one of
the reviewers of this manuscript. - This re-
viewer also indicated that ionic contrast media
are no longer administered intravenously, but
used instead to fill lumens (i.e., bladders, fis-
tulas, etc.).
6.6.4 Angiography. Methiodal sodium, so-
dium iodomethamate, and iodopyracet were
the earliest water-soluble organic iodine com-
pounds available for angiography (10). From
the incidence. of patient reactions and from
laboratory investigations, it became evident
that these compounds were not entirely suit-
able for angiography use. In about 1950, sub-
stituted triiodobenzoic acid derivatives were
introduced as contrast agents (219,490). Ace-
trizoate was the first of the series introduced.
followed by diprotrizoate and then by diatrizo-
ate, iothalamate, metrizoate, iodamide, and
ioxitalamic acid. Acetrizoate and diprotrizoate
were found to have certain toxic effects but
diatrizoate, iothalamate, and metrizoate were
better tolerated and have found extensive use
in angiography (490). Iodamide and ioxita-
lamic acid are widely used in Europe. Benton
et al. (854) found that meglumine calcium dia-
trizoate is superior to meglumine diatrizoate
and meglumine sodium diatrizoate. In 1970,
dimers and trimers of contrast agents and
nondissociable compounds
- were introduced
for angiographic use (9). Newer agents such as
iocarmic acid (bisiothalamate), iozomic acid,
and tris(iothalamate), as well as the nondisso-
ciable metrizamide, have fewer side effects
and are better tolerated. Metrizamide, the
first-generation nonionic contrast medium, .
has been the choice for cardioangiography and
coronary angiography (855). The low osmola-
lity ionic ioxaglate and the second-generation
nonionic iopamidol and iohexol are better tol-
erated than the ionic contrast media. with no
measurable clinical disadvantage in diagnos-
tic and therapeutic cardiac radiology, particu-
larly in high risk patients (856). Coronary an-
giography with iopamidol is less painful and
-
has fewer deleterious effects on myocardium
metabolism and cardiovascular system than
the ionic media (857- 859).
6.6.5 Cholangiography. Intravenous chol-
angiography was introduced in 1954 as a ra-
diographic technique in the diagnosis of bili-
ary tract disease and is practiced in cases in
which conditions causing obstruction to pas-
sage and resorption are present and prevent
the use of oral cholecystography (737). The
sole contrast agent used in the procedure in
the United States is iodipamide, which is

available as sodium and meglumine salts. The
difference between the two salt forms is in the
volume of the dosage required for examina-
tion, with the volume of meglumine salt less
than that of sodium salt. Adverse reactions
after a single bolus injection are reduced if the
contrast medium is administered by slow-in-
fusion technique.
Many other cholangiographic agents are
available. Brismar et al. (860) studied ioglyc-
amide or ioglycamic acid in relation to iodip-
amide. Ioglycamide is widely used in Europe
and has an excellent contrast property for ex-
aminations of bile ducts after intravenous in-
jection but offers no advantage over iodip-
amide in examinations of gall bladder. An
intravenous cholangiographic agent, iodox-
amic acid, introduced in 1973, apparently has
lower toxicity (190, 861).
Cholangiography agents are mostly dimers
of triiodobenzoates, such as iodipamide, iogly-
carnide, iodoxamate, iotroxate, and iosul-
amide, with high water solubility and high bil-
iary excretion (862). Iosulamide with a fully
substituted triiodophenyl ring was found ex-
perimentally to cause less severe reaction and
more rapid opacification than iodipamide
(323).
6.6.6 Cholecystography. Graham et al. in
1924 (863) obtained the first cholecystogram
in dogs with tetrabromophenolphthalein. Tet-
raiodophenolphthalein was used for cholecys-
tography until 1943, when iodoalphionic acid
was introduced (175), which produced better
radiographic visualization of the gall bladder
with fewer toxic reactions. Its use was super-
seded by iopanoic acid, a substituted triiodo-
benzoic acid introduced in 1951 (864). Io-
panoic acid is better tolerated and has a
greater opacity than iodoalphionic acid. The
N-butyryl derivative of iopanoic acid, tyro-
panoate, was introduced in 1963 and was
found to be more readily absorbed from the GI
tract than iopanoic acid. This compound, in
addition to rapid oral absorption, gives excel-
lent opacification of the gall bladder under
conditions of clinical use and has a low inci-
dence of side effects. Opacification with tyro-
panoate occurs with diagnostic quality on the
4-h f l m and the 8-h film in 64 and 78% of the
patients examined, respectively (865,866).
Radiopaques
A number of substituted 2,4,6-triiodoben-
zoic acid derivatives were introduced as chole-
cystographic agents. Among them, bunamio-
dyl and iophenoxic acid were later withdrawn
by the manufacturers because bunamiodyl
caused renal shutdown and iophenoxic acid
caused high protein-bound iodine values per-
sisting over many years.
Ipodate, introduced in 1961, can yield visu-
alization of both gall bladder and bile ducts.
Tyropanoate, iopanoic acid, and ipodate are
widely used in oral cholecystography. They
show about the same efficacy but differ in the
intensity of opacification, the frequency of dim
and absent shadows, and the frequency of side
effects. Russell and Frederick (867) compared
these three agents but failed to demonstrate
the superiority of any one of them. Iocetamic
acid was favorably compared with and pre-
ferred to the above three agents, but skin re-
actions were reported in a few cases (775,868,
869).
6.6.7 Myelography. Oil-soluble and water-
soluble contrast agents have been used in my-
elography. These agents are administered in-
tracisternally. The absorption of oily and
water-soluble contrast media from the sub-
arachnoid space is completely different. The
oily media remain in the subarachnoid space
for years after injection, whereas the water-
soluble media are eliminated within a few days
(799).
The water-soluble iodopyracet was intro-
duced for myelography in 1931 (870). Because
of the extreme irritant effects as well as occa-
sional production of some long-term disabili-
ties, it never gained wide use. Iodized oil Lipi-
odol was used for myelography with less
irritation. Its aftereffects were avoided if the
oily medium was removed by aspiration from
the spinal canal (871). Iophendylate was intro-
duced by Ramsey et al. (872) for myelography
in 1944 and proved to be an excellent contrast
medium for the entire spinal canal and the
basal cisterns. Like the iodized oil, it had to be
aspirated to avoid aftereffects (873). Arach-
noiditis did develop in some cases in the sub-
arachnoid space because of the presence of re-
sidual iophendylate.
Iothalamate meglumine was used for my-
elography in 1964. Melartin et al. (656) showed
7 Miscellaneous Agents
that meglumine and iothalamate ions are less
neurotoxic than sodium and diatrizoate ions.
The irritating effect of meglumine iothalamate
is so slight that its use in myelography re-
quires no spinal anesthesia (682). Iocarmic
acid (i.e., dimerized iothalamate), with a
higher iodine content and a lower toxicity
than those of iothalamate, has been used in
myelography (214, 874). Both iocarmic acid
and iothalamate tend to be spasmogenic, caus-
ing clonic convulsions or muscular twitchings
in the leg (875). Nonionic, water-soluble me-
trizamide was introduced for myelography in
1973 (876). It is less irritating than available
myelographic agents and is capable of visual-
izingsmall structures such as nerve roots, root
pockets, and blood vessels. Nonionic, water-
soluble iopamidol causes fewer and less severe
adverse reactions particularly in terms of behav-
iorial changes and compares favorably to metri-
zarnide (857). Iopamidol also induces less dis-
comfort to the patients in cerebral angiography
than iothalamate and diatrizoate (877).
6.6.8 Urography. Contrast agents rapidly
excreted by the kidney are used in excretion
urography. Iopax, or Uroselectan, was first in-
troduced for urography in Germany in 1929.
Many other agents such as methiodal sodium,
hippuran (sodium oiodohippurate), sodium io-
domethamate, and iodopyracet were also in-
troduced. These agents were the earliest avail-
able, and iodopyracet was the most widely
used (490).
With the introduction of modern contrast
agents beginning in about 1950, urography
has been conducted by use of the water-solu-
ble acetrizoate, diatrizoate, iodamide, iodip-
amide, iothalamate. and metrizoate. These
agents, because of their higher radiopacity
and better tolerance, have superseded the ear-
lier agents, although their individual toxicities
vary. Among these, diatrizoate is the least
toxic and the most favored in urography (490).
Contrast agents cause osmotic diuresis.
Studies show that sodium diatrizoate is ex-
creted in higher
- concentration in the urine
than is meglumine diatrizoate because of re-
sorption of sodium ions in the renal tubules.
Benness (790, 878) reported a difference in
roentgenographic quality in pyelograms in fa-
vor of sodium contrast agents.
Dimeric and trimeric contrast agents with
more iodine atoms per molecule improve the
opacity of urine by their high iodine concen-
tration and cause a decrease in osmotic diure-
sis. In sheep, these new large molecules pro-
duced smaller urinary solute output, less
diuresis, and higher urinary concentration
than the same parameters of diatrizoate (879,
880, 809). These agents are clinically useful.
Urine iodine concentration, after the injec-
tion of nonionic metrizamide. is about twice as
high as after sodium diatrizoate injection. Gol-
man et al. (795) reported that during the peri-
ods of ureteric stasis, metrizamide was ex-
creted faster than diatrizoate. Nonionic
iopamidol
* reduces the incidence of adverse re-
actions in excretory urography and produces
equal quality urograms with less iodine than
diatrizoate (881).
7 MISCELLANEOUS AGENTS
7.1 Perfluoroctyl Bromide
In addition to organic iodinated compounds,
perfluorocarbons are also potential contrast
agents. The least toxic one among them is per-
fluoroctyl bromide (C,F,,Br), which has been
introduced by Long et al. (882-884) for bron-
chography, myelography, splenography, lym-
phangiography, and special gastrointestinal
studies requiring small volumes of nontoxic
radiopaques.
Perfluoroctyl bromide (PFOB) has a high
degree of chemical and physical stability and
nonreactivity, a biologic inertness comparable
to that of Teflon, a high oxygen solubility, and
a low surface tension. Liquids with low surface
tension wet surfaces readily and flow freely
into tiny folds and orifices. This property in a
contrast agent improves the quality of the
roentgenogram. PFOB has a radiopacity
about 50% of that of iothalamate, and may be
administered as neat liquid or emulsions.
Emulsions in normal saline can be prepared
with the aid of the emulsifying agent Pluronic
F-68. Perfluorocarbon emulsions have a non-
Newtonian viscosity that can be varied over a
very wide range by changing perfluorocarbon
and surfactant concentrations and emulsify-
ing technique (882,883).
572
Perfluoroctyl bromide, when given trache-
ally either as neat liquid or as a 10:l emulsion,
was cleared roentgenologically from the lung
within 24 h. Evaporation and mucociliary
clearance play an important role in the elimi-
nation of this radiopaque from the lungs.
When the tissue samples were analyzed by gas
chromatography, approximately 1% of the
neat liquid and 8% of the emulsion were found
still remainingin the tissue. The lungs had the
largest amount, followed by the intestine, ad-
ipose tissue, and lymph nodes. When injected
intrathecally into the dog, a dose of 0.5-1.0
mL/kg caused no toxic manifestation. It is
much less irritating than iodophendylate to
the arachnoid tissue.
Perfluoroctyl bromide is especially useful
roentgenographically for patients who are al-
lergic to iodine compounds and who have had
meningitis before myelography. Emulsions of
PFOB have been investigated for contrast en-
hancement of tumors (8451, lymph channels
and nodes in animals (840), and as an arthro-
graphic contrast agent in the knee joints of
dogs and a human cadaver (885). The PFOB
emulsion is less viscous and easier to inject
than Ethiodol for lymphography. The high
sensitivity of computed tomography to mini-
mal density differences between tissues allows
PFOB to be experimented in the rabbits for
contrast enhancement of malignant neo-
plasma (8861, reticuloendothelial systems
(liver, spleen, and macrophages), and VX2 tu-
mors (887), and in CT imaging of septic and
aseptic arthritis (888). PFOB produces in CT
prolonged opacification of the blood pool and
subsequent selective enhancement of liver
and spleen in the pig for days (889). The fluo-
rocarbon causes no acute hemodynamic effect
in comparison to ionic contrast media. The
LD,, value for intravenously administered
48% PFOB emulsions in rats was found to be
approximately 25 or 30 mL/kg, and the agent
is eliminated as vapor from the organism
through the lungs and skin (887). PFOB may
activate the immune system and stimulate
macrophage migration to the sites of infection
and neoplasia. Information on details of the
whole-body toxicity of PFOB and on the mech-
anisms of vacuole formation in macrophages
has been scanty.
Radiopaques
7.2 lodinated Particulate Suspensions
After bolus injection, water-soluble contrast
media, routinely used in angiogrphy, are rap-
idly distributed throughout the intravascular
space and diffuse across the capillary wall into
the interstitial space. A brief time window ex-
ists for imaging the differential contrast be-
tween organs and adjacent tissues before non-
specific total body opacification occurs. In
comparison, particulate contrast media are
not hyperosmolar, remain longer in the intra-
vascular space, and are specifically targeted to
organs, such as blood pool, liver, and spleen
(890). Particulate contrast media, when ad-
ministered in the form of emulsions, lipo-
somes, microcapsules, and nanocrystals, for
example, exert no osmotic pressure and are
rapidly taken up by the macrophages and the
Kupffer cells in the reticuloendothelial (RE)
system and accumulate in the liver and spleen.
The RE system is saturable, and overflow from
high doses will persist in the circulation. Par-
ticulate agents produce high contrast between
normal and pathologic tissues at a fraction of
the dose required of conventional agents and
maintain the density difference for CT imag-
ing over prolonged periods of time. The ad-
verse effects of the particulates, including the
clearance, the effect of long-term retention,
Kupffer cell activation, disturbances in'the
microcirculation of the liver (8911, and vacu-
oles in the RE cells (892), have yet to be fur-
ther studied. Violante and Fisher (893) dis-
cussed the complexities and problems in
designing and formulation of RE-selective
contrast media. Later advances in producing
liposomes and encapsulated vesicles entrap-
ping water-soluble iodinated contrast media
and other particulate suspensions are briefly
mentioned below:
7.2.1 Liposomes. Liposomes are vesicles
consisting of one or more bilayers of lipid en-
capsulating an aqueous core, carrying water-
soluble contrast agents. The weight ratio of
iodine to lipid (I/L ratio) is a measure of the
amount of iodine per unit weight of lipid deliv-
ered to the target organ (894). For hepatic en-
hancement in CT this ratio needs to exceed 1:l
(895). The leakage and the I/L ratio of lipo-
somes depend to a large extent on the lipid
7 Miscellaneous Agents
components, the microemulsification process,
and the nature of water-soluble contrast
agent. Methods for producing contrast-carry-
ing liposomes may be categorized as ( 1 ) the
microemulsification method using a high pres-
sure microfluidizer (894, 896, 897), (2)the de-
hydration-rehydration method (891,898), and
(3) the reverse-phase evaporation method
(899,901).
Cheng et al. (894) produced liposomes by
microemulsification through use of a high
pressure microfluidizer from a contrast agent
and a mixture of egg phosphatidylcholine,
phosphatidylglycerol, and cholesterol in the
molar ratio of 0.4:0.1:0.5. The liposomes car-
rying nonionic contrast agents have higher en-
trapment and weight ratio than those carry-
ing ionic contrast agents; the decreasing order
is as follows: iotrolan > metrizamide > io-
hex01 > iopamidol > ioxaglate > diatrizoate >
iodipamide. For diatrizoate the mean I/L ratio
was 0.062 + 0.01 mg/mg and the entrapment
value, 0.12 ? 0.02 Llmole. The weight ratio for
nonionic contrast agents increased signifi-
cantly in the liposomes with increasing initial
concentrations of the contrast agent. The
leakage rate of encapsulated diatrizoate was
over 20%in physiological saline. Encapsulated
iohexol gave greater and more prolonged
opacification of the liver and spleen than plain
iohexol. Maximum contrast was reached
within 30 min after injection and maintained
for more than 60 min. The results indicated
that osmotic pressure, charge, viscosity, and
lipophilicity of contrast media might influence
the encapsulation process.
Leakage of water-soluble contrast agents
from the encapsulated liposomes can be elim-
inated, using an interdigited lipid phase con-
sisting of hydrogenated soy phosphatidylcho-
line or distearoyl phosphatidylcholine to
produce "interdigitation-fusion" (IF) vesicles.
Janoff et al. (897) found that the IF vesicles
carrying iotrolan, ioversol, ioparnidol, ioxa-
glate, and diatrizoate indeed have higher I/L
ratios varying from 5.5 to 8.9, and vesicle sizes
ranging from 1 to 5 pm in diameter. Leakage
by incubation of the liposomes in uitro with
fetal calf serum at 37°C for 4-24 h showed that
iotrolan and ioxaglate liposomes retained al-
most all of the iodine, whereas iopamidol and
diatrizoate liposomes lost about 50 and 80% of
the encapsulated iodine, respectively. In dogs
and rats, 1 h after injection at dose levels rang-
ing from 25 to 250 mg Ikg, the liver retained
an average of 93.7%of the injected 1251-labeled
iotrolan IF-vesicles and the blood retained less
than 5% of the dose (895). At 24 h the initial
dose in the liver fell to about 10% and de-
creased thereafter with a half-life of about 6 h.
By 115 days, 0.2% of the initial dose still re-
mained in the liver. The high liver uptake was
attributed to less lipid in the IF-vesicles so
that the RE system was not prematurely
saturated.
Krause et al. (899,900) studied the charac-
terization of iopromide liposomes, produced
from a lipid mixture of phosphatidyl choline,
cholesterol, and stearic acid at a molar ratio of
4:5:1 by a dehydration-rehydration method.
The liposome had an average diameter of 0.5
+ 0.1 pm. Encapsulation efficiency was be-
tween 30 and 40%, with an iodine-lipid (IIL)
ratio of approximately 1. The iopromide-car-
rying liposomes had a leakage of 4.6%in rabbit
plasma and 9.2% in human plasma in the 0- to
4-h period and a leakage of 9% in rabbit
plasma and 20% in human plasma in the 0- to
24-h period. The LD,, value of iopromide lipo-
somes was approximately 3 g I/ kg in rat and
mouse. The pharmacokinetic parameters of
the liposomes in rat, such as total clearance .
and terminal half-life, were dose dependent.
The terminal half-life in blood was increased
from 0.8 h for a 250 mg I k g dose to 2.9 h for
1000 mg I k g dose, suggesting a longer circu-
lation of liposomes after higher doses. When
the liposomes were given to rats at a dose of
250 mg Ilkg by mouth, recoveries (% of dose)
from excretion in 0-72 h were approximately
93% (feces) and 3% (urine); by subcutaneous
injection, recoveries in 0-48 h: 7% (feces),
61% (urine), and 31% (injection site); by intra-
muscular injection, recoveries in 0-72 h: 9%
(feces),69% (urine), and 6% (injection site). In
day 7, a total of 0.36%of the administered dose
was recovered in organs and tissues. Thus, io-
promide-carrying liposomes proved to be sta-
ble and highly tolerable vesicles for the target-
ing to specific organs in animal model. Fifteen
minutes after intravenous injection of 100 mg
Ikg, rat blood showed the highest proportions
of iodine, followed by the liver, the spleen, and
the kidney. Significant proportions of iodine
574
were found only in the liver and spleen, after
total clearance from the blood and the kid-
neys. In the rabbits with VX2 carcinoma, the
iopromide liposome showed strong contrast
enhancement of the liver. In the dog, inter-
digital injection of iopromide liposomes en-
hanced the contrast of ipsilateral lymph nodes
(899). Ioxaglate-carrying liposomes with a
lipid component consisting of egg phosphati-
dylcholine alpha-tocopherol had a mean lipo-
somal diameter of 220 nm and an encapsula-
tion efficiency of 85% and at larger doses
(2250 mg I k g bw), showed a 10-fold greater
enhancement for the spleen than for the liver
and a sustained intravascular contrast en-
hancement of the aorta (902).
Petersein et al. (903) evaluated in healthy
rabbits two new liposomal contrast agents
aimed at the reticuloendothelial system for
liver CT: BR2 and BR21 (from Bracco Re-
search SA, Geneva, Switzerland). Both are
suspensions of iomeprol-containing liposomes
in an iomeprol solution; BR2 has twice the li-
posome concentration of BR21, that is, 40 vs.
20 mg lipid/mL, at an iodine content of 260 vs.
320 mg IImL, respectively. The liposomes are
0.4 mm unilamellar vesicles, made of a phos-
pholipid bilayer surrounding an aqueous
phase. The membranes consist of phospholip-
ids (phosphatidyl choline and dipalmitoyl
phosphatidic acid) and cholesterol in a 2:1 mo-
lar ratio. The authors studied the time course
of contrast enhancement in liver CT with the
liposomal contrast agents, by use of extracel-
lular iomeprol at 300 mg IImL as the control.
In healthy rabbits, at doses of 1.5 mLkg or
greater, the two liposomal agents induce a sig-
nificantly stronger and more prolonged en-
hancement of the liver than that of the extra-
cellular iomeprol and provide a larger imaging
window for optimizing CT examinations of the
liver. Dick et al. (904) evaluated a new con-
trast agent, liposomal iodixanol (LI), made of a
1:1mixture of 10% glucose and iodixanol(50%
encapsulated iodixanol: 1 mL of that mixture
contained 50 mg encapsulated iodine) for ex-
aminations of pyrogenic liver abscess in a rab-
bit model by CT. The experimental group of
animals received the LI at a dose of 200 mg
Ikg, and the control group received iopentol
at a dose of 600 mg Ikg. Results showed that
the LI exceeds the extracellular iopentol in
Radiopaques
overall abscess contrast and duration of the
diagnostic interval, in that the liposomal io-
dixanol gives higher hepatic vessel contrast
and better abscess localization.
Activity of encapsulated liposomes is
closely related to the method of preparation.
Musu et al. (890) prepared large unilamellar
vesicles (0.3-1 p) carrying iopamidol from
phosphatidylcholine and dipalmitoylphospha-
tidic acid in a 9:l ratio and iopamidol solution
(300 mg IImL) and extruded the vesicles
through a polycarbonate membrane of differ-
ent pore sizes (0.8-2.0 p). Extrusion above the
transition temperature (75°C) of the lipids re-
duced the average size and size distribution of
the vesicles and increased their I/L ratio. Dis-
tribution studies of extruded and unextruded
iopamidol-carrying liposomes in rats showed
that extruded liposomes gave higher spleen
uptake than did unextruded, whereas the liver
uptake was comparable. Lung entrapment
was significant with unextruded but almost
eliminated with extruded liposomes.
After intravenous injection conventional li-
posomes are rapidly taken up by cells of the
mononuclear phagocytic system (MPS). The
inclusion of glycolipids and hydrophilic poly-
mers in the liposome membrane modifies the
surface characteristics to evade the MPS sys-
tem and increases the liposomal circulation
time (905). Sache et al. (906) prepared ioprd-
mide-containing liposomes by the continuous
high pressure extrusion method, and used the
liposomes without prior removal of unencap-
sulated contrast agent for surface modifica-
tion. The liposome membranes were made
from soy phosphatidylcholine (SPC), choles-
terol (CH), and soy phosphatidylglycerol
(SPG) in a 6:3:1 (SPCICHISPG) molar ratio,
and with the original lipid concentration at
120 mg/g total suspension. The liposome
membranes were modified by inclusion of lipid
derivatives of polyethylene glycol (PEG) by
coating, simply carried out by mixing the pre-
formed iopromide-containing liposomes with
5 mol % of either of the two coating agents
(DSPE-PEG2000 or CHHS-PEG2000)for 16 h
at room temperature with stirring. The
DSPE-PEG coating increased the mean diam-
eter of the vesicles to approximately 200 nm,
probably attributable to aggregation and fu-
sion of the vesicles, and decreased the zeta po-
7 Miscellaneous Agents
tential of the negative surface charge. The sta-
bility of the unmodified and surface-modified
iopromide liposomes in human plasma was de-
termined by equilibrium dialysis of a lipo-
some/plasma mixture against the respective
plasma to be stable over a period of 6 h. The
biodistribution of modified and unmodified io-
promide liposomes was studied in rats, and no
significant differences in blood concentration
could be found 1 h after injection between dif-
ferent formulations at a dose of 250 mg I/kg
body weight, corresponding to 500 mg lipidkg.
Computed tomographic images were studied
in rabbits. The unmodified and DSEP-PEG-
modified liposomes displayed prolonged blood
concentration with CT density differences
above 70 HU units (aorta) for up to 20 min and
proved to be useful for CT imaging, displaying
favorable imaging properties.
Liposomes may also serve as a vehicle for
carrying an oil-soluble contrast agent such as
Ethiodol to the liver (907). To form Ethiodol
liposomes, a chloroform solution of Ethiodol
and a mixture of egg phosphatidylcholine and
phosphatidic acid in a molar ratio of 7:l and a
chloroform solution of Ethiodol are mixed in a
proportion of 3:Z, followed by solvent evapora-
tion and addition of water with stirring and
sonication. The Ethiodol is contained within
the liposomes, probably in the hydrophobic re-
gion. The liposomes have diameters of 0.015 to
0.2 p and show rapid liver uptake in the rab-
bits and long retention times. Unlike the nor-
mal liver tissue the tumor tissue in the rabbits
with implanted VX2 carcinoma accumulates
no Ethiodol. The dose of Ethiodol liposomes
needed for contrast enhancement of the liver
is about 1113th of that required of water-solu-
ble diatrizoate.
Caride et al. (908)incorporated brominated
phosphatidylcholine with or without choles-
terol into brominated radiopaque liposomes.
The liposomes were 1-5 p in diameter and
showed contrast enhancement of the liver in
the dog 1 h after intravenous injection. A few
hours after injection brominated liposomes
were found inside the hepatocytes. Because
the bromine atom is not as effective as the
iodine atom in attenuating X-rays, a corre-
spondingly large dose of the brominated lipo-
somes had to be administered for imaging. No
information on chemical characterization of
brominated phosphatidylcholine or its fate
and biotransformation was given.
Yang et al. (909) used poly(~,~-lactide), a
polymer with molecular weight in the range of
5000-50,000 Da, for microcapsulation of ethyl
esters of iopanoate and diatrizoate and Ethio-
do1 by a solvent evaporation method. The mi-
crocapsules were about l pm in diameter. Par-
ticles of this diameter or smaller can safely
traverse the pulmonary capillary bed to be
available for the liver and Kupffer cell phago-
cytosis. In viva microscopic examination re-
vealed the activity of Kupffer cells phagocytiz-
ing the microcapsules. These particles were
essentially macrophage-imaging agents. Mi-
crocapsules of all three contrast media in-
creased the density of the liver in the rabbits.
Maximum opacification of the liver paren-
chyma appeared 15-30 min after injection,
leaving any focal hepatic lesions as defects.
7.2.2 Emulsions. Ethiodol may also be
emulsified by mixing with a small amount of
phospholipid as emulsifying agent (910). The
oil droplets of size 2 to 3 microns in the emul-
sion are rapidly and efficiently taken up by the
RE system of the liver. The imaging quality of
ethiodol emulsions is comparable to that of
ethiodol liposomes.
Microemulsions of a series of polyiodinated
triglycerides (ITG), labeled with iodine-125
and processed in a microfluidizer, were inves-
tigated for their contrast enhancement of the
liver and the tumor in normal and tumor-
bearing (Walker 256) rats, in rabbits bearing
VX2 carcinoma, and in normal dogs in CT
(911). The mean particle diameter of micro-
emulsions was less than 300 nm. These prep-
arations were stable and autoclavable. Thirty
minutes after intravenous injection, from 66
to 78% of the injected dose remained in the
liver of the rats. After 3 h the liver still re-
tained from 46% to 93% of the dose. At dose
levels ranging from 20 to 70 mg Ikg, the in-
crease in density was reported to be about 40
HU in the rats. In a female pig, the contrast
enhancement within 1 h of injection was 90
HU. The liver uptake of ITGs was partially
dependent on the formulation vehicle, but the
metabolism and clearance from the liver were

dependent on the chemical structure and the
alkyl chain length (911).
7.2.3 Particulates. Particulates are pre-
pared from insoluble derivatives of contrast
agents, finely milled to uniform size, and sus-
pended in water in the presence of small
amounts of surfactants and stabilizers. These
particles have sizes between 200 and 400 nm
in diameter and are referred to as nanopar-
ticles. Nanoparticles can be formulated as
blood-pool and liver-spleen CT contrast agents
for injection at concentrations of 15-20% (wl
v), corresponding to 89-118 mg IImL. The in-
soluble ethyl esters of ionic contrast media in
nanoparticles will be taken up by the RE sys-
tem upon injection and hydrolyzed by ester-
ases into ethanol and water-soluble ionic con-
trast agent and excreted. Rubin et al. (912)
prepared nanoparticles from the water-insol-
uble diatrizoate ethyl ester and investigated
the effect of different types of surfactants on
contrast enhancement of aorta, liver, and
spleen in the rabbits, and used iohexol as com-
parator. In the presence of a high molecular
weight nonionic polymeric surfactant, the
nanoparticles produced excellent and pro-
longed enhancement of aorta and vena cava,
but in the presence of a low molecular weight
anionic nonpolymeric surfactant, the nano-
particles markedly opacified liver and spleen.
This difference in targeting to different organs
was attributed to the shielding of the nanopar-
ticles from opsonization by the high molecular
weight nonionic polymeric surfactant, causing
a decrease in the uptake by the RE system.
Gazelle et al. (913) tested more than 50 insol-
uble derivatives of diatrizoic acid, iothalamic
acid, urokonic acid (acetrizoic acid), and met-
rizoic acid, formulated as nanocrystals for
blood-pool and liver-spleen imaging. Most of
these agents demonstrated either blood- or
liver-dominant enhancement patterns in nor-
mal rabbits and rabbits with VX2 carcinoma.
Some of the nanoparticles gave improved
liver-to-lesion contrast compared to that of io-
hexol. The chemical identity of these insoluble
derivatives was not divulged.
Violante et al. (914,915) prepared particles
of iothalamate ethyl ester with a diameter of 2
1 pm by injecting a solution of ethyl
iothalamate in dimethylformamide at a rate of
Radiopaques
3 mL1min into a 5% aqueous solution of poly-
vinylpyrrolidone (PVP), chilled to 0-2°C and
circulated at a rate of 1000 mL/min. The par-
ticles precipitated out from solution as white
suspensions after being warmed at 40°C for 30
min and were centrifuged at 3000g for 5 min,
repeatedly washed with water, and resus-
pended in saline for injection. These particles
were stable in normal saline and showed no
particle aggregation when mixed with rat, rab-
bit, dog, or human serum but formed aggre-
gates up to 80 pm in diameter in plasma. It
was found that fibrinogen interacts with the
particles, forming aggregates. Aggregation
was prevented by preincubating the particles
in human serum albumin before introducing
them into plasma. The authors also found that
in solution, iothalamate has a higher intrave-
nous LD,, value in mice than that of iodipam-
ide (13-19 g/kg versus approximately 4 g/kg),
whereas as nanoparticles, iothalamate ethyl
ester has a lower intravenous LD,, by rapid
injection in mice than that of iodipamide ethyl
ester (550 mg I/kg versus 1200 mg I/kg). Lee et
al. (916) noted that in computed tomographic
portography bolus injections of iodipamide
ethyl ester particles were consistent in detect-
ing all the lesions in pathologic liver in a ca-
nine model. This demonstrated that chole-
graphic agents could be used to produce
particulate contrast agents.
Li et al. (917, 918) synthesized ioxilan car-
bonate by reaction of ioxilan with carbonyldi-
imidazole in dimethyl sulfoxide. The reaction
is specific for nonionic contrast media, protect-
ing all the hydroxyl groups in the molecule and
rendering it water insoluble. The reaction is
given on the following page.
The ioxilan carbonate particles were pre-
pared as a contrast medium by solvent extract/
evaporation method. The preparation in-
volved emulsification of a methylene chloride
solution of the carbonate, removal of solvent,
and washing and sizing the particles. The io-
dine content of the particles was 45%. The av-
erage diameter of the ioxilan carbonate parti-
cles was 1.1 pm, 95% of them ranging from 0.6
and 2.0 pm. Electron microscopy showed the
particle surface to be smooth, and the particles
showed practically no aggregation when
mixed with rat plasma. The LD,, value for the
ioxilan carbonate particles was 1.4 g I/kg for
7 Miscellaneous Agents

cH3co
\
NI
HZC
'GII
CONHCHzCHzOH
Carbonyldiimldazole
Dimethyl sulfoxide
*

I
CHOH
I
CHzOH

Ioxilan Ioxilan Carbonate

male and 1.2 g I/kg for female mice, corre-

spondingto a particle mass of 3.1 and 2.6 &g
body weight, respectively. Liver attenuation
enhancement was 38 HU after intravenous
injection of 200 mg I/kg of the carbonate
particles in normal rabbits and rabbits with
implanted VX2 carcinoma. Attenuation en-
hancement was dose dependent and increased
with the dose. At a dose of 270 mg I/kg the
attenuation enhancement was 110 HU. Liver
enhancement reached maximum approxi-
mately 30 min after injection, and the spleen
enhancement was many times higher than
that of the liver. Tumors in the liver of rabbits
bearing VX2 carcinomas were detected. Iox-
ilan carbonate particles were rapidly cleared
from the blood. Opacification of the gallblad-
der and the kidneys was observed, indicating
that the particles and their degradation prod-
ucts were excreted through hepatic and uri-
nary pathways.
7.3 iodinated Polymers
Soluble starch treated with ethylene oxide and
iodinated with N-iodosuccinimide yields 6-io-
doethylated starch (IES). The repeating unit
of IES is shown in the following column.
Sako et al. (919) prepared two iodinated
polymers, IES-200 and IES-40. IES-200 has an
average molecular weight of 200,000 Da, an
iodine content of 12.5%,a water solubility of
0.8 g/mL, and a CT number of 1510 HU.
IES-40 has an average molecular weight less
than 40,000 Da, an iodine content of 20%, a
water solubility of 1.0 g/mL, and a CT number
of 1900 HU. Both preparations have biocom-
patible viscosity and osmolality, show no acute
toxicity in mice at dose levels of 5 and 10 g/kg
body weight, and also no serological toxicity at
5 g k g in rabbits. On intravascular injection
the iodinated ethylated starch is retained in
the blood for a considerable length of time
with minimum leakage from the capillary
wall. On intraparenchymal injection the iodin-
ated starch opacifies the regional lymph
nodes. The iodinated soluble starch is a poten-
tial contrast agent for blood-pool and vascular
bed opacification and for indirect lymphogra-
P~Y.
Other polymers have also been investi-
gated as blood-pool contrast agents. Revel et
al. (920) used an iodinated polymer consisting
of a carboxymethyldextran with a triiodinated
benzoic acid substituent. The iodinated poly-
mer had a mean molecular weight of 32,000
Da, ranging from lo3 to lo6 Da and contained
24 mg I per 100 mg of powder. An intravenous

solution for injection had an iodine concentra-
tion of 8.7% with an osmolality of 560 mOsml
kg. This iodinated polymer enhanced the con-
trast differentially, up to 10 min, between
normally perfused and ischemic liver and
showed blood-flow differences at the capillary
level in normal rabbits and rabbits suffering -
segmental portal ischemia. The polymeric ma-
terial, because of its high molecular weight,
was confined to the vascular space and was
able to achieve vascular enhancement with
about one-fourth of the iodine concentration
required for diatrizoate, a water-soluble con-
trast agent. Doucet et al. (921) also reported
the use of an iodinated carboxymethyl dextran
containing an unidentified triiodinated ben-
zoic acid substituent group of a molecular
weight of about 32,000 Da as a blood-pool X-
ray contrast agent. Lautrou et al. (922) stud-
ied the pharmacokinetics of iodinated polymer
P509 (mol. wt., 47,000) as a blood-imaging
product and compared it with ioxaglate. The
iodinated polymer P509 showed a consider-
ably higher plasma concentration, a lower uri-
nary excretion, a considerably lower biliary
excretion, and an elimination half-time twice
as long as that of ioxaglate. These studies dem-
onstrate that iodinated polymers are poten-
tially useful blood-pool contrast agents.
The above-mentioned blood-pool contrast
media are polymers into which are grafted io-
dinated groups, ranging widely in molecular
size and prone to be antigenic. As an alterna-
tive, Sovak et al. (923) proposed and synthe-
sized blood-pool radiopaque polymers by copo-
lymerization of triiodinated isophthalmic acid
species containing acrylic or methacrylic acid
groups with a nonradiopaque component con-
sisting of acrylic or methacrylic acids, with or
without their hydroxylated amides to form
polymers of about 50,000 in molecular weight.
These polymers with suitable physicochemical
properties, intravenous tolerance, and the ca-
pability of being excreted were selected and
further linked by biodegradable bis-acrylic
linkages to form large polymers, in excess of
50,000 in molecular weight. These polymers
were uniform in size as compared to the
grafted radiopaque polymers and were biode-
gradable and excretable after degradation.
The molecular configuration of these poly-
mers is such that they contain extended hy-
Radiopaques
drophobic regions and triiodophenyl rings
with carboxylic acid groups, which is contrary
to the design theory so successfully applied to
the design of nonionic radiopaque agents. The
authors remarked that in view of this observa-
tion, the development of radiopaque polymers
may need to proceed on a trial-and-error basis.
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CHAPTER ELEVEN

Microarrays and Gene

~ x ~ r e s s i oProfiling
n Applied to
Drug Research
BRAD WINDLE
Department of Medicinal Chemistry
Center for Bioelectronics, Biosensors and Biochips (C3B)
Virginia Commonwealth University
Richmond, Virginia

ANTHONY GUISEPPI-ELIE
Department of Chemical Engineering
Center for Bioelectronics, Biosensors and Biochips (C3B)
Virginia Commonwealth University
Richmond, Virginia

Contents
1 Introduction, 600
2 DNA Microarray Technology and Methods in .
Gene Expression Analysis, 600
2.1 DNA Microarray Fabrication, 600
2.2Applications of DNA Microarrays, 601
2.3 Interpretation and Bioinformatics of
Microarray Data, 605
3 Applications of Gene Expression Profiling in
Drug Research, 606
3.1 Characterizing Cells by Gene Expression
Profiling, 606
3.2 Gene Expression and Drug Response, 608
3.3 Pathways and Targets, 610
3.4 Simulating Effects of Drugs through
Genomics, 612
3.5 Molecular Toxicology, 614
3.6 Bioinformatics Meets Chemoinformatics, 614
4 Concluding Remarks, 614
5 Acknowledgments, 615

Burger's Medicinal Chemistry and Drug Discovery

Sixth Edition, Volume 4: Autocoids, Diagnostics,
and Drugs from New Biology
Edited by Donald J. Abraham
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc.
 Microarrays and Gene Expression Profiling Applied to Drug Research
1 INTRODUCTION
The sequencing of the human genome repre-
sented a milestone accomplishment of the
20th century. The 23 pairs of human chromo-
somes that comprise almost three billion base
pairs of deoxyribonucleic acid (DNA) have
now been established to contain approxi-
mately 38,000 genes that encode some
60,000-80,000 different proteins (l,2). Tradi-
tional analyses of genes and proteins are gen-
erally directed to one gene or protein at a time.
This leisurely approach cannot match the pace
of modern genomics and makes it difficult, al-
most impossible, to unearth complex interac-
tions among genes or proteins. Traditional
methods cannot readily reveal multigene rela-
tionships, the complex temporal symphony of
gene action, or reveal the "whole picture" of
gene functions and interactions. The study of
the interactions among so many cellular fac-
tors requires the development of complex an-
alytical tools and the use of sophisticated com-
puter methods in the management and
analysis of the data derived from such tools.
The start of the 21 century is witnessing
the embryonic development of a new bioana-
lytical technology for the analysis of gene ex-
pression and genotyping-DNA microarrays.
DNA arrays using oligonucleotides were first
developed for a hybridization-based DNA se-
quencing method (3). DNA microarrays have
more recently been developed as tools for
studyinggene expression (4).DNA microarray
technology translates what the cell is display-
ing at the genomic level into interpretable
data. This technology enables researchers to
obtain instantaneous snapshots of the expres-
sion levels of all genes. It also offers the poten-
tial for understanding complex regulatory
pathways, deriving the distinguishing genetic
identities of individuals, and discovering and
understanding the molecular basis for disease
states.
Modern genomics and DNA microarrays
have been described as tools for revolutioniz-
ing drug development (5). However, promises
of the genomics-driven revolution in drug re-
search come with few details. How does know-
ing the sequence of all genes in our cells help
us discover or develop drugs? In the fields of
drug discovery, drug design, and drug develop-
ment, microarrays are becoming the instru-
ments for displaying complex drug interac-
tions in the cells at the genomic and proteomic
level. This chapter describes principles of
DNA microarray technology and basic analy-
sis of DNA microarray data, followed by dis-
cussions of recent ventures in applying DNA
microarray technology to drug research.
2 D N A MICROARRAY TECHNOLOGY
A N D METHODS IN GENE EXPRESSION
ANALYSIS
2.1 D N A Microarray Fabrication
DNA microarrays fall under the category of
DNA chips. The term DNA chip is used to gen-
erally describe a solid platform cut from a
larger substrate by dicing (hence chip) that
incorporates a molecular component derived
from deoxyribonucleic acid, ribonucleic acid,
or biomimetic versions of these genetic mole-
cules. An array used in biological studies is an
orderly arrangement of known biological enti-
ties displayed in two dimensions on a flat sur-
face. These entities may be (1)proteins, such
as with antibody microarrays, (2)nucleic acids
used to study gene expression or gene copy
number in cells, or (3) tissues where the tis-
sue-specific level of expression of a protein or
gene is being explored. There can also be com-
binatorial microarrays aimed at materials de-
velopment. DNA microarrays are thus far the
most developed and widely used of the mi-
croarray devices. The array of DNA material
may be printed on glass, supported hydrogel,
nylon, or metal substrates. In its simplest
manifestation, the only requirement is that
the substrate be flat, smooth, chemically uni-
form, and provide a physicochemical means
for the attachment of the several DNA se-
quences. In more complex manifestations, the
substrate may present three dimensionally
such as a substrate-supported hydrogel layer,
a microporous membrane, an etched-rough-
ened surface, or a channel-bearing substrate.
2 DNA Microarray Technology and Methods in Gene Expression Analysis
Figure 11.1. Pin tool for spotting DNA on a glass
slide. Panel a shows a four-pin device acquiring
DNA solution from a microtiter well plate. The DNA
solution is printed onto a glass slide in panel b. The
DNA identities, well positions, and the spot posi-
tions are determined in a pattern program used by
the spotter arrayer.
Currently, the most widely used substrate is
glass, with dimensions of the familiar 3.0" x
1.O" microscope slide.
There are two general platforms for DNA
microarray fabrication. The most versatile of
these platforms uses high-speed robotics
(spotter arrayer) with DNA solutions. In a
spotter arrayer, precision robots are combined
with nanoliter liquid transfer capability. The
nanoliter liquid transfer device may be of two
generic types: contact or non-contact transfer
of liquid to create the spots of the microarray.
Contact methods include pin-tools and quills.
Pin-tools are typically pencil-like devices with
laser sharpened titanium tips. Quills are sim-
ilar but possess a laser-drilled capillary run-
ning from the tip up the center of the shaft.
Both exploit the surface tension forces of wa-
ter to create a uniform drop of DNA solution
that is transferred to the substrate on physical
contact (Fig. 11.1). Non-contact methods use
electromechanical actuation to propel a fixed
volume of the DNA solution to the surface.
This method can use inkjet heads, micro-sole-
noid valve dispensers, or piezoelectrically
driven liquid dispensers. The DNA being ar-
rayed can be bound to the substrate through a
variety of fixation chemistries. Pin-tool robots
can print greater than 25,000 spots on a glass
microscope slide, given a feature size as small
as 100 pm with 200-pm center-to-center spac-
ing.
The second platform for DNA microarray
fabrication involves controlled in situ synthe-
sis of oligonucleotides (on-chip). This technol-
ogy, implemented by Affymetrix in the manu-
facture of GeneChips, uses photolithographic
601
masks to expose selected spots to light, result-
ing in deprotection of reactive groups needed
for nucleotide coupling (Fig.11.2). Each nucle-
otide added is itself protected, requiring light
exposure to deprotect for subsequent addition
of the next nucleotide. A different mask is
used for each nucleotide added, and the pro-
cess is repeated until each spot has oligonucle-
otides with a specific DNA sequence. The oli-
gonucleotide length for GeneChips is 25 bases.
Using this photolithographic technique, the
synthesis of 25-base oligonucleotides requires
100 chemical synthetic steps and can poten-
tially give rise to 39,635 (254) unique probe
sequences. Because of the high precision of the
photolithography process, a feature size of 20
pm is obtained, allowing the production of
chips with as many as 400,000 spots.
2.2 Applications of D N A Microarrays
DNA microarrays can be used in a variety of
experimental protocols to reveal different
types of data about the genome. The type of
data desired determines the kind of DNA that
is arrayed. Cloned genomic DNA can be ar-
rayed for the analysis of DNA or gene copy
number in cells, for example, in tumor cells
that may have undergone gene loss or ampli-
fication (6). Oligonucleotide arrays have been
particularly useful in DNA sequence analysis
and single nucleotide polymorphism (SNP)
scanning (7). The arraying of both oligonucle-
otides and cDNA sequences has been exten-
sively used in analysis of gene expression. Ar-
rays allow the parallel determination of the
expression level for all genes in a cell, thus
providing a complete gene expression profile.
Two general applications for DNA microar-
rays in gene expression analysis are to com-
pare gene expression patterns between cells of
different tissues and to compare gene expres-
sion patterns in a single cell type under differ-
ent conditions. An example of the latter appli-
cation is the comparison of gene expression
profiles between drug-treated cells and un-
treated control cells. In contrast, gene expres-
sion profiling of different tissues does not al-
low the luxury of control cells for comparison,
and thus the profiles are only compared to
each other. Table 11.1 lists a number of stud-
ies that have employed DNA microarray tech-
602 Microarrays and Gene Expression Profiling Applied to Drug Research

Figure 11.2. Photolithographic process for on-chip

synthesis of oligonucleotides. A: the steps in this pro-
cess in two cycles of nucleotide addition. A litho-
graphic mask and light source are shown in panel a
that results in exposure of specific spots on the mi-
croarray chip. The light activates reactive groups on
the chip such that nucleotide coupling can occur only
on the specific spots as shown in panel b. The nucle-
otide added is shown by green blocks and is thymi-
dine for this example. Panel c shows a new mask
allowing exposure of new spots to light in a second
round of light activation of reactive groups. Panel d
shows the next round of nucleotide addition in which
the red blocks represent the next nucleotide, for ex-
ample, adenosine. This process is repeated until all
spots have the desired DNA sequence. B and C show
schematic illustrations of the many steps in the
solid-phase synthesis of the oligonucleotides (A) us-
ing MeNPOC chemistry (B). In this example, 4
masks are needed to produce the first base of each
oligonucleotide, and 80 masks are needed to produce
a 20-base oligo chip. See color insert.

nology for gene expression profiling. To date, The expression level of a cell's genes, and in
the field of cancer research has gained the turn its proteins, is the major determinant of
most from using microarray technology, with cell type and function. One traditional method
other fields rapidly catching up. for quantitating the gene expression level or
 Microarrays and Gene Expression Profiling Applied to Drug Research

Figure 11.2. Photolithographic process for on-chip

synthesis of oligonucleotides. A: the steps in this pro-
cess in two cycles of nucleotide addition. A litho-
graphic mask and light source are shown in panel a
that results in exposure of specific
. spots
. on the mi-
croarray chip. The light activates reactive groups on
the chip such that nucleotide coupling can occur only
on the specific spots as shown in panel b. The nucle-
otide added is shown by green blocks and is thymi-
dine for this example.
- Panel c shows a new mask
allowing exposure of new spots to light in a second
round of light activation of reactive groups. Panel d
shows the next round of nucleotide addition in which
the red blocks represent the next nucleotide, for ex-
ample, adenosine. This process is repeated until all
spots have the desired DNA sequence. B and C show
schematic illustrations of the many steps in the
solid-phase synthesis of the oligonucleotides (A) us-
ing M ~ N P O Cchemistry ( B ) . - I ~this example, 4
masks are needed to vroduce the first base of each
A

oligonucleotide, and 80 masks are needed to produce

a 20-base oligo chip. See color insert.

nology for gene expression profiling. To date, The expression level of a cell's genes, and in
the field of cancer research has gained the turn its proteins, is the major determinant of
most from using microarray technology, with cell type and function. One traditional method
other fields rapidly catching up. for quantitating the gene expression level or
2 DNA Microarray Technofogy and Mefhods in Gene Expression Analysis 603

C T C G T C T T
A G A G G A G C
T A C A T C A A
C C G C A G C T
G A T A C T A G
T T C C A A G G

Figure 11.2. (Continued.)

concentration of a specific mRNA within a cell
is Northern blot hybridization. This method
involves fixing total mRNA (target sequence)
from cells of interest to a membrane substrate
and hybridizing with a labeled DNA probe spe-
cific for the gene of interest. The relative level
of mRNA in the cells is calculated by normal-
izing the label's signal to the signal from an
internal control mRNA, such as a housekeep-
ing gene detected by a separate hybridization.
Using this method, the relative gene expres-
sion level for a single gene can be determined
for multiple cell samples (e.g., 10-20 samples).
Whereas microarray analysis of gene ex-
pression employs similar steps as Northern
blot hybridization, it is radically different in
principle. The major difference is that the
roles of the nucleic acid bound to the substrate
and the labeled nucleic acid in the hybridiza-
tion solution are reversed for microarrays. In
microarray analysis, the DNA probe is fixed to
the substrate with each spot on the array con-
 Microarrays and Gene Expression Profiling Applied to Drug Research
Figure 11.2I. (Continued.)
taining DNA specific for a single gene. The
hybridization solution for microarrays con-
tains target cDNA sequences produced from a
cell's mRNA, as opposed to a probe sequence
specific for a single gene. Thus, whereas a
Northern blot measures the expression of a
single gene for many samples, the microarray
measures the expression of thousands of genes
for a single sample.
Spotted arrays are routinely used to simul-
taneously analyze gene expression in two cell
samples using one hybridization. In many
cases, one of the two samples represents a con-
trol cell, providing an internally controlled
reference for each spot hybridization. In con-
Table 11.1 Applications for Gene
Expression Profiling
Studies References
Categorization of tissues and
tumors
Identification of transcriptional
regulatory pathways
Disease gene identification
Drug characterization
Identification of drug interactions
and mechanism of action
Identification of diagnostic and
prognostic markers
Toxicologic responses
Predicting therapeutic efficacy
trast, GeneChip arrays are hybridized with
only a single sample. The analysis of two sam-
ples in one hybridization is made possible by
separately incorporating into each sample's
cDNA sequences one of two optically distinct
fluorophores (e.g., Cy3 and Cy5). For example,
mRNA extracted from cells treated with drug
will be labeled during cDNA synthesis with
Cy3, whereas the mRNA extracted from the
untreated cells will be labeled with Cy5. The
separately labeled cDNA sequences are mixed
and hybridized to the DNA microarray chip
(Fig 11.3). The number of molecules of probe
DNA in each microarray spot is in far excess of
the number of labeled cDNA molecules in so-
lution, and thus the two labeled cDNAs do not
compete, but hybridize independently to their
specific DNA spots. The amount of labeled
cDNA bound to a particular gene probe or spot
is therefore indicative of the level of expres-
sion for that gene's mRNA.
The amount of Cy3 and Cy5 label bound to
the microarray is quantitated by a fluores-
cence scanner that generates a grayscale im-
age for each label. The two images are typi-
cally pseudo-colored, for example green for
Cy3 and red for Cy5. The intensities of the two
images are normalized, and the images are
overlaid to produce a single image that dis-
plays the relative expression of each gene
based on hue (Fig. 11.4). For studies using
2 DNA Microarray Technology and Methods in Gene Expression Analysis
Figure 11.3. Hybridization of two differentially la-
beled cDNA sequences to arrayed DNA. This dia-
gram shows a single array spot with probe DNA
strands (in gray) bound to the chip. cDNA sequences
from two different cell samples, one labeled with
Cy3 (green) and one labeled with Cy5 (red), are hy-
bridized to the spotted DNA. See color insert.
GeneChips, two images from separate hybrid-
izations are overlaid, relying on the chips' pre-
cision and reproducibility for proper image
alignment.
If the abundance of a particular mRNA
from the drug-treated cells is equal to that
from the control cells, then the spot specific for
that mRNA will be yellow. The spot will ap-
pear green if the mRNA is present in greater
abundance in drug-treated cells than in con-
trol cells, and red if the mRNA from the drug-
treated cells is lower. A potential anomaly in
the analysis is caused by differences in how
the two fluorescent labels are incorporated in
the sample DNAs, which can result in the ap-
pearance of differences in genes expression be-
tween the two samples. It is typical to repeat
hybridizations with the two fluorescent labels
reversed for the samples labeled to help verify
and validate legitimate differences in gene ex-
pression.
Low intensity spots suggest an overall low
level of gene expression for a gene, although
other factors can also contribute to the inten-
sity. These factors include labeling efficiency
for each mRNA, the size of each mRNA, and
the hybridization kinetics for each DNA se-
quence. Hybridization kinetics would not be
an issue if the hybridization reaction was al-
lowed to go to completion. However, hybrid-
izations typically are carried out overnight
and not necessarily until all possible DNA hy-
brids are formed. The image in Fig. 11.4 allows
a visual and qualitative analysis of relative
gene expression, but it is the extraction of spot
intensities from the images that provides the
data for quantitative analysis.
2.3 Interpretation and Bioinformatics
of Microarray Data
The raw data obtained from microarray anal-
yses rarely remain raw for long. The data are
simmered in a series of transformations
needed to address basic questions of gene ex-
pression. Typical transformations include ,
background subtraction, normalization, ratio
calculations, and log transformation. The nor-
malization is required because the scale for
fluorescence emissions from different fluoro-
phores are rarely equivalent. In addition, the
scale for signal from the same fluorescent label
Figure 11.4. A spotted DNA array with two-
color detection of hybridization. An example of a
spotted DNA array (16 X 20 elements) is shown
after hybridization with two differentially la-
beled cDNA preparations, Cy3 (pseudo-colored
green) and Cy5 (pseudo-colored red). The over-
laying of the green and red images produces the
image shown. The hue of each spot, ranging from
green to red, indicates the relative expression
level for the gene specific for each spot (image
courtesy Packard Biochip Technologies). See
color insert.
606 Microarrays and Gene Expression Profiling Applied to Drug Research
may not be the same from experiment to ex-
periment. The validity of the normalization
and ratio calculations relies on the bulk of the
genes exhibiting the same levels of gene ex-
pression in each cell analyzed. If 90%of a cell's
genes were to increase threefold in expression
level under a specific condition, this event
would not be detected. Instead, the interpre-
tation would be that expression of the unaf-
fected 10% of genes decreased approximately
threefold compared with the control cells.
The ratio calculation is well suited for two-
label hybridization experiments, as shown in
Fig. 11.4, where there is an internal control for
hybridization. The ratio calculation provides
an indication of induction or repression of
gene expression relative to the control. The
distribution and variance of the ratio data are
also usually analyzed to determine thresholds
for values significantly higher or lower than
one, although in many cases this threshold is
arbitrarily chosen and is typically at the level
of a twofold change. The log transformation is
used to bring the distribution closer to a nor-
mal distribution for statistical analyses. Be-
cause of the high number of genes analyzed
and the few observations or replicate experi-
ments, it can be difficult to demonstrate sta-
tistical significance for small changes in gene
expression.
A common goal in studying gene expression
from microarray data is to identify groups of
genes, cells, or conditions displaying similar
patterns. A widely used tool for reaching this
goal is cluster analysis (8). Cluster analysis
organizes items into clusters based on how
close the items are to each other using a dis-
tance or similarity calculation between items.
Hierarchical clustering not only allows clus-
tering of items but clustering of clusters and is
usually presented in the form of a hierarchical
tree diagram or dendrogram. An example of
this analysis is shown in Fig. 11.5 in which 60
cell lines were clustered based on the gene ex-
pression levels of 548 selected genes. Most of
the cell lines cluster based on tissue origin.
The lengths of the horizontal lines connecting
the cells relates to the distance between cell
line data points or similarity. The two cell
lines, MDA-MB-435 and MDA-N, have the
shortest distance between their data points or
the greatest similarity, and thus have the
shortest horizontal lines connecting them. A
typical measure of similarity is based on the
Pearson correlation coefficient, which was
used in the clustering in Fig. 11.4. The signif-
icance of these clusters will be discussed later
in this chapter. An overview of the basic meth-
ods used in microarray data analysis is pre-
sented by Quackenbush (9).
3 APPLICATIONS OF GENE EXPRESSION
PROFILING I N DRUG RESEARCH
3.1 Characterizing Cells by Gene Expression
Profiling
Virtually all human gene sequences have been
arrayed and are being used to create gene ex-
pression profiles in a wide range of cell types.
Instead of the noted phrase, "you are what you
eat", we might now say "you are what you
express." The gene expression profile of a cell
currently represents the most precise defini-
tion of what a cell is, what it does, and what it
can do. Of course, a detailed protein expres-
sion profile would theoretically be superior,
but this must await advancement in the field
of proteomics (10).
Gene expression profiling has been used
with great success to categorize or classify cell
types, particularly cancers. For example, this
approach has been employed to characterize
lymphoma cells and identify subtypes of lym-
phomas from patients with diffuse large B-cell
lymphoma. These cells express similar gene
expression patterns because of their lympho-
cytic lineage, and they exhibit only subtle dif-
ferences in patterns. Alizadeh et al. (11)iden-
tified two distinct subtypes of lymphomas
using spotted arrays containing -18,000
genes (Lymphochip), which were otherwise
essentially indistinguishable. These two sub-
types had subtle differences in gene expres-
sion profiles that were in common with two
different B-cell lineages, suggesting that they
represent two distinct diseases. Furthermore,
the two subtypes were found to exhibit a dif-
ferential response to therapy, that is, patients
with one subtype showed significantly better
survival than patients with the other subtype,
while on the standard anthracycline-based
therapy. It's important to note that most
genes were expressed at comparable levels in
3 Applications of Gene Expression Profiling in Drug Research

BRE MDA-MB-435
ERE MDA-N

718 Melanomas UACC-257

MEL MALME-3M
SK-MEL-2
MEL SK-MEL-5

CNS Cancers

NSC
BRE MDA-MB-231
ERE ADR RES
OVA OVCAR-8
NSC HOP-62
REN SNI2C
788-0
TK-f 0
RXF-393
Renal REN UO-32
ACHN
CAKI-I
A498

CCRF-CEM
MOLT-4
HL-60
616 Leukemias SR

OVA
OVA
OVA
PRO
PRO
OVA
OVA
BRE
BRE
COL
COL
COL
Colon Cancers COL
COL
COL
COL
NSC
NC1-H23
NCI-H522 1
A549 -
1
EKVX
NCLH460
NCI-H226

Figure 11.5. Cluster analysis of the NCI 60 cell lines. Gene expression data from the Ross et al. (12)
study was filtered to contain only genes with complete data for all cell lines and genes with the highest
variance across cell lines. Only 548 genes were selected for this analysis based on genes with variance
of greater than twice the variance for the entire dataset. The cluster distance or similarity calculation
was based on the Pearson correlation coefficient, and clusters were linked by average linkage using
the software SPSS. Most cell lines can be seen to cluster based on tissue of origin, shown by the labels
on the left and the fraction of the total number of cell lines for each tissue. The abbreviations for
tissue types are as follows: BRE, breast cancers; CNS, central nervous system cancers; COL, colon
cancers; LEU, leukemias; MEL, melanomas; NSC, non-small cell lung cancers; OVA, ovarian cancers;
PRO, prostate cancers; REN, renal cancers. See color insert.
608 Microarrays and Gene Expression Profiling Applied to Drug Research
both lymphoma subtypes, and only a subset of
genes displayed differential expression pat-
terns that contributed to the distinction be-
tween the subtypes. The gene expression lev-
els of this subset of genes may potentially be
useful as a diagnostic tool for distinguishing
between the two subtypes and predicting re-
sponse to therapy.
A variety of tumor specimens have now
been characterized by gene expression profil-
ing (11-16). In some cases, the gene expres-
sion profiles have confirmed distinctions be-
tween tumor types that were already evident
by histologic or cytogenetic analysis. In other
studies, even finer distinctions between tu-
mors types, not apparent histologically, have
been made possible by gene expression profil-
ing. For example, Hedenfalk et al. (17) identi-
fied specific gene expression patterns that cor-
relate with BRCAl and BRCA2 mutations in
hereditary breast cancer. These findings are
complicated by the fact that the BRCAl and
BRCA2 mutations correlated with other fac-
tors such as tumor grade, mitotic index, and
presence or absence of hormone receptors.
Many of the genes expression patterns in
these cells should also reflect these differences
in processes and pathways responsible for tu-
mor characteristics. The power of the microar-
ray/bioinformatics approach is that it offers
the potential to identify gene expression pat-
terns that correlate with BRCAl or BRCA2
mutations independent of hormone receptor
status, as well as patterns that correlate with
hormone receptor status independent of the
BRCAlI BRCA2 mutation status.
Gene expression profiling can also provide
insight into a tumor's tissue or cell type of
origin. Gene expression profiles were used to
determine cell origin for a panel of 60 human
cancer cell lines (NCI 60 cells) representing
cancers of breast, prostate, lung, central ner-
vous system, leukemia, melanoma, renal, co-
lon, and ovarian origins (12). Gene expression
levels were determined in each cell line for
over 9000 genes using spotted DNA arrays.
Cluster analysis of the 60 cell lines was per-
formed using approximately 1000 genes that
showed the highest variance across cell lines.
The clustering generally grouped cell lines of
the same type together. One exception was the
breast cancer cell lines that were scattered
among the other cell line groups. Figure 11.5
shows a hierarchical tree diagram of a cluster
analysis we performed on the data from Ross
et al. (12) in which a slightly different variance
calculation and cluster distance calculation
were used. Again, clusters of cell lines gener-
ally coincided with the tissue of origin, similar
to the published analysis, but with an im-
provement in the clustering of lung cancers.
The breast cancer cell lines retained a hetero-
geneous distribution pattern, with some clus-
tering with the melanoma, CNS cancer, and
colon cancer clusters. These studies illustrate
the use of gene expression profiling for deter-
mining tissue origin and suggest how gene ex-
pression profiling could be useful in defining
cell behavior.
In addition to cell lines with similar gene
expression profiles being clustered, the genes
can also be clustered based on expression pat-
terns across the 60 cell lines. Cluster analysis
of the genes from the 60 cell lines revealed
clusters of genes that are part of the same bi-
ological process or pathway (12). For example,
one cluster represented genes that are highly
expressed in melanomas and involved in mel-
anocyte-specific biology. Other clusters con-
tained genes encoding proteins that are in-
volved in cell cycle progression, RNA splicing,
and drug metabolism. It is hypothesized from
these studies that groups of genes relating to
basic cellular processes such as cell prolifera-
tion, cell morphology, or drug response could
be identified by this type of gene expression
analysis. The identification of previously un-
identified genes in these clusters may help elu-
cidate the functions of the encoded proteins.
Much of this advancement will come from the
development of new algorithms for analyzing
the data and detecting patterns amidst the
considerable noise of a cell's repertoire of ex-
pressed genes.
3.2 Cene Expression and Drug Response
The difference between a compound and an
agent is dictated by the cell just as the differ-
ence between an agent and a drug is dictated
by the patient. However, at this point, we
would like to make a simplification of terms in
which a compound, an agent, or a drug will
3 Applications of Gene Expression Profiling in Drug Research
merely be referred to as a drug even though a
compound or agent may not be of known med-
ical value.
The NCI 60 cell lines have been screened
for sensitivity to a ~ 7 0 , 0 0 0drug library, re-
sulting in a wealth of data for identification of
patterns and relationships between drugs
(18).The search for drugs within this library
that have sensitivity patterns related to a drug
of interest can identify drugs with similar
structure and or mechanism of action [See the
COMPARE program (19)l. While this data is
of enormous use. there is even more data to be
obtained relating to the genes and proteins
expressed by the cells that dictate how the
cells respond to the drugs.
The microarray- data for the NCI 60 cell
lines can be viewed as a source of ~ 9 0 0 0
marker genes for potential correlation with bi-
ological properties, including sensitivity to an-
ti-cancer drugs. Scherf et al. (20) explored the
relationship between basal gene expression
patterns and drug sensitivity in these cell lines
using drug activity data for the ~ 7 0 , 0 0 0
drugs. We can begin to understand the chal-
lenges in bioinformatics when we consider the
enormous task of melding the data for these
three distinct types of variables: 60 cell lines,
9000 genes, and 70,000 drugs, and looking for
relationships and patterns. One straightfor-
ward approach for finding relationships in this
data is to focus on genes and drugs for which
there is a considerable amount of additional
information. For example, the activity of 5-flu-
oruracil (5-FU), a known thymidylate syn-
thetase inhibitor, was found by Scherf et al.
(20) to have a significant negative correlation
with expression of the gene encoding dihydro-
pyrimidine dehydrogenase (DPYD). DPYD is
involved in uracil and thymine degradation as
well as in 5-FU degradation, and thus high
expression of the DPYD gene is consistent
with resistance to 5-FU. Staunton et al. (21)
carried this approach further identifying sub-
sets of genes whose expression levels in the
NCI 60 cell lines showed good prediction of
sensitivity for certain drugs. However, it was
not readily apparent what role the identified
genes or their proteins played in the sensitiv-
ity to the drugs. This level of understanding is
likely to require a very detailed knowledge of
the pathways with which each drug interacts.
609
One approach Scherf et al. (20) used for
identifying relationships between drug re-
sponse and gene expression involved cluster-
ing the drugs based on their correlation with
expression of each gene. A distance calculation
between each drug pair was performed using
correlation coefficients between the drugs and
each gene. This clustering placed drugs in
groups sharing similar relationships between
drug response and gene expression rather
than drug response alone. The results were
compared with the cluster analysis, using only
drug response data. Drug clusters from the
drug response data tended to contain drugs
with similar mechanisms of action, such as an-
timetabolites or tubulin inhibitors. The drug
cluster analysis based on gene-drug correla-
tions also tended to group drugs based on
mechanisms of action, but clustered certain
drugs in groups with distinctly different
mechanisms of action. For example, the topo-
isomerase I1 inhibitor, etoposide, clustered
with other topoisomerase I1 inhibitors using
drug response data, but clustered with DNA
alkylators when the drug-gene correlation
data was used. These results could reflect a
potential difference in mechanisms of action
or drug metabolism between etoposide and the
other topoisomerase I1 inhibitors.
The application of gene expression profil- .
ing is beginning to pave the way for improved
treatment of patients using existing therapeu-
tic agents. First, knowledge of a tumor's gene
expression profile may greatly facilitate tumor
type identification, leading to more accurate
predictions of tumor response to therapies.
Second, gene expression analysis can poten-
tially reveal patterns that predict therapeutic
response or help identify proteins that play in
a specific role in response to therapy. This ap-
proach was explored by Kihara et al. (22) in a
study of esophageal cancer patients treated
with 5-FU and cisplatin. The expression pro-
file of ~ 9 0 0 genes
0 was determined for patient
tumors before treatment, and a subset of
genes with predictive value for subsequent
therapeutic response was identified.
Another promising application of gene ex-
pression profiling is in clinical drug develop-
ment. For example, a drug that seems to have
a poor overall response rate in clinical trials
may actually have a significant activity in a
610 Microarrays and Gene Expression Profiling Applied to Drug Research
subset of patients whose tumors may share
particular gene expression patterns. The abil-
ity to preselect a narrower target group of can-
didates for clinical trial could lead not only to
improved trial outcome but also to a cost-sav-
ing reduction in trial size. Until now, the num-
ber of markers used in this way has been lim-
ited, but microarray data is opening a
floodgate in the identification of potential
markers for drug response.
The use of gene expression profiling to im-
prove therapeutics overlaps with a relatively
new field of study, pharmacogenomics. Phar-
macogenomics is the study of the genomic or
genetic basis for variation in response to drugs
observed between patients. This field of study
attempts to define and understand differences
in drug response at the patient level to enable
tailoring of therapeutics to individuals. Much
of the field has focused on the study of DNA
polymorphisms, particularly single nucleotide
polymorphisms (SNPs),and their relationship
with drug response. The analysis of SNPs also
relies heavily on the use of DNA microarray
technology. Information about the field of
pharmacogenomics and innovations are pre-
sented in this volume by Puckett et al. (23).
3.3 Pathways and Targets
The cellular response to a drug depends on
both the drug's specific and non-specific inter-
actions in the cell. Whereas a particular drug
may be known to inhibit a protein target, in-
hibition of that target may represent only a
fraction of what the drug is doing in the cell. In
fact, the primary mechanism of action may not
even involve the supposed target. The chal-
lenge then, is to record and interpret all as-
pects of the cellular response to a drug, rather
than a single parameter, such as cell growth.
Gene expression profiling offers a much
deeper probe into the cellular response to a
drug. Two general strategies for use of gene
expression profiling are emerging in drug re-
search; the first is involves determining the
basal gene expression profile of a cell as a
means of obtaining predictive information
about a cell's drug response, as described
above. The second strategy involves determin-
ing the change in a cell's gene expression pro-
file in response to drug treatment. The drug
response profile adds an entirely new dimen-
sion to understanding of the relationship be-
tween gene expression and drug response, be-
cause it reflects the targets and pathways with
which the drug- interacts and the downstream
effects of these interactions on gene expres-
sion. The cell's response to a drug at the
genomic and proteomic level potentially pro-
vides a detailed fingerprint reflecting the full
range of effects of the drug on the cell.
This concept is illustrated using a simple
protein kinase signal transduction pathway as
an example (Fig. 11.6). This generic protein
kinase cascade translates a signal from a
membrane receptor to the nucleus resulting in
increased expression of genes A, B, and C, as
well as the repressed expression of genes X, Y,
and Z. This pathway also has a branch such
that PK2 interacts with other pathways and
can affect the expression of other genes (genes
E, F, and G ) . These interactions or branches
may be cell-type specific, and thus the choice of
cells for analysis can dictate the genes af-
fected. The goal is to explore drugs that will
block the high expression of genes A, B, and C.
A panel of drugs (panel1)previously known to
block PK1 is first considered. The effect of
each drug in panel 1 is the desired repression
of genes A, B, and C as well as the de-repressed
expression of genes X, Y, and Z. Each of these
PK1 inhibitors also affects the expression of
genes associated with the branching path-
ways. In addition, some of these inhibitors
may interact with proteins not connected to
the targeted pathway, such as kinases in un-
related pathways. The additional genes af-
fected by the PK1 inhibitors can be very dis-
tinct for each inhibitor and can provide part of
the fingerprint that distinguishes each inhibi-
tor from the others. This part of the finger-
print may also reveal side interactions of the
PK1 inhibitors that were not previously
known or anticipated. The same approach can
potentially be applied to cases in which little is
known about a drug of interest. Clues about
the drug's interactions could be deduced from
its similarity in gene expression changes with
other drugs- for which there is more informa-
tion regarding cellular interactions and mech-
anisms.
This protein kinase pathway also shows
how inhibition of other *proteins in the same
pathway could give rise to similar changes in
3 Applications of Gene Expression Profiling in Drug Research
Genes
Figure 11.6. A generic protein kinase pathway for
cell signaling. This diagram shows a ligand binding
to a cell surface receptor, thereby activating a signal
transduction cascade. The ligand-induced activa-
tion of the receptor leads to activation of protein
kinase PK1, which then activates PK2, which in
turn activates PK3. PK3 modifies transcription fac-
tors in the nucleus that lead to high expression of
genes A, B, and C, and low expression of genes X, Y,
and Z. PK2 also interacts with another pathway that
leads to high expression of genes E, F, and G.
downstream gene expression. For example,
consider a drug speculated to have some gen-
eral specificity for protein kinases. This drug
has a similar effect on the expression of genes
A, B, and C and X, Y, and Z, but it has no effect
on the expression of genes E, F, and G. We
might deduce from this result that the drug
blocks a step downstream of the branch at
PK2, such as PK3. This drug would be worth
611
pursuing because of its increased specificity
compared with drugs in panel 1. Therefore,
similarity in gene expression fingerprints may
identify a common pathway with which the
drugs interact, rather than a common protein.
Thus, the definition of a target may be broad-
ened from a single cellular component to a
pathway, allowing for the identification of ad-
ditional drugs of interest with dissimilar
structure and mechanism of action. For a well-
understood pathway, the gene expression pro-
file could potentially pinpoint the specific step
in the pathway inhibited by a drug.
This approach to characterizing kinase in-
hibitors was used by Gray et al. (241, in a study
of gene expression changes induced in yeast
cells by purine analogs that target human cy-
clin-dependent kinase 2 (CDK.2) and Saccha-
romyces cereuisiae cyclin-dependent kinase
cdc28p. Virtually the entire complement of
yeast genes (6200 genes) was monitored for
changes in expression in response to two CDK
active site inhibitors with dissimilar struc-
tures and a related purine analog with poor
CDK inhibition. More than 194 genes showed
significant changes in expression, with both
CDK active site inhibitors eliciting similar
changes in expression for 63 of these genes.
The majority of the affected genes were up-
regulated in expression. The drug with poor
CDK inhibition gave rise to changes in expres-
sion of only two genes, consistent with its in-
ability to block cdc28p or other protein ki-
nases.
A number of cell cycle-related genes were
found to be affected by the two CDK inhibi-
tors, including several cyclin and histone
genes, consistent with previous data suggest-
ing that these genes are regulated by cdc28p.
Another interesting class of genes affected by
the CDK inhibitors contained genes involved
in phosphate metabolism. This is significant
because, in addition to cdc28p, the CDK inhib-
itors also inhibit pho85p, a closely related ki-
nase that is involved in the regulation of phos-
phate levels. This illustrates the potential for
gene expression profiling to reveal multiple
drug-target interactions within the cell.
Other genes of interest that were up-regulated
included genes encoding various heat-shock
proteins and proteins involved in drug extru-
sion pump systems. Induction of heat-shock
612 Microarrays and Gene Expression Profiling Applied to Drug Research
genes suggests the cells exhibited a stress re-
sponse to the CDK inhibitors. However, the
induction of these stress response genes may
not necessarily reflect a direct response to
cdc28p inhibition, but rather may represent a
response to the cascade of events stemming
from the perturbation of the cell cycle. The
change in expression of genes for drug pump-
associated proteins suggests possible mecha-
nisms of resistance and could explain differ-
ences in growth response to the drugs. It is
important to note that not all changes in gene
expression are likely to be informative. It's ex-
pected that many of the genes affected in this
type of experiment will merely represent ge-
neric responses to gross cellular perturbations
and will be affected by a large number of
drugs, revealing very little information about
an individual drug's unique properties.
The genes affected concordantly by both
CDK inhibitors are not the only genes of inter-
est. The majority of the affected genes by ei-
ther inhibitor were unique to each drug,
suggesting that the two CDK inhibitors inter-
acted with more unique proteins than com-
mon proteins. Two basic questions arise from
this analysis: how does one identify the af-
fected genes specific to cdc28p inhibition (or
any specific target perturbation) and how does
one identify the pathways associated with the
other affected genes? Both questions can be
addressed through the use of genomics, as dis-
cussed in the next section.
The potential of gene expression profiling
for drug classification is also illustrated by a
study of adrenergic receptor-interactive drugs
(25). Twenty drugs known to be agonists, an-
tagonists, or both for a- and p-adrenergic re-
ceptors were used as a test set for the analysis
of gene expression profile responses. Normal
human aortic smooth muscle cells, which ex-
press both a- and P-adrenergic receptors, were
used as the model cells for the treatments. Of
the 6000 human genes analyzed for expres-
sion, a subset of 75 genes was selected for clus-
ter analysis after filtering out genes with low
or no response to drug and genes with the low-
est variance. Cluster analysis of the drugs
based on patterns of induced gene expression
changes cleanly separated the agonists from
the antagonists. Although the number of
drugs analyzed was low, there was also some
indication of drug clustering based on the spe-
cific receptor with which the drugs interact.
As the use of gene expression profiling of
drug response expands, we can envision the
development of reference libraries in which
gene expression patterns are equated to
classes of drugs, structures, and mechanisms
of action. As new drugs are investigated, they
could be mapped to drugs in the library to elu-
cidate mechanisms, identify more specific
drugs, or obtain leads with specific cellular ef-
fects.
3.4 Simulating Effects of Drugs through
Cenomics
By coupling yeast genetics with microarray
analysis, one can identify the transcriptionally
regulated processes associated with each gene.
This approach has been successfully used in
studying the mitogen-activated protein kinase
signaling pathway, allowing identification of
new associated regulatory circuits (26, 27).
The mutation of a target protein is also a way
to block its activity and simulate the inhibi-
tion of the target by a drug. In the study of the
CDK inhibitors by Gray et al. (24) described in
the previous section, the gene expression pro-
file for a yeast cdc28 mutant was compared
with gene expression profiles of wild-type
yeast cells treated with the cdc28p inhibitors.
The changes in gene expression patterns re-
sulting from cdc28 mutation had significant
overlap with those found for the two CDK in-
hibitors. This analysis therefore helped to val-
idate the responses observed, indicating that
the CDK inhibitors were in fact targeting
cdc28p in the cell. Not surprisingly, there were
several differences in gene expression changes
induced by the CDK inhibitors and the cdc28
mutation, consistent with the prediction that
the CDK inhibitors would be less specific than
cdc28 mutation. However, the difference
could also be caused in part by the use of a
temperature-sensitive conditional mutant of
cdc28, which was required because loss of
cdc28p is lethal. The change in temperature
therefore adds another variable with which to
contend. Whereas each method of disrupting
the target seems to have its own unique ef-
fects, each unique effect can theoretically be
determined and isolated within the gene ex-
 pression data, thus allowing complex biologi-
cal processes to be unraveled and understood.
A more global approach to characterizing
gene expression patterns in yeast mutants
was undertaken by Hughes et al. (28).Almost
300 specific mutants were analyzed for the ex-
pression of virtually all yeast genes. This da-
tabase provides a resource for analyzing regu-
latory pathways associated with the target
genes and identification of genes associated
with cellular processes. The database also pro-
vides a reference library for mapping gene mu-
tants and drugs to the pathways they perturb.
As with drug-induced gene expression pat-
terns or fingerprints, mutant gene response
patterns can be used to match mutants with
similar fingerprints. For example, mutants
known to affect sterol synthesis cluster to-
gether based on their gene expression profile.
The expressed genes involved in sterol synthe-
sis also cluster based on their expression
across the ~ 3 0 mutants.
0 The clustering of a
mutant for a gene of unknown function with
mutants of genes with known function sug-
gests by association a function for the un-
known gene. This was demonstrated with a
mutant for a gene that clustered with mutants
of genes involved in sterol biosynthesis. Fur-
ther investigation into this gene provided evi-
dence of its role in sterol biosynthesis.
Identifying similarities between a gene ex-
pression fingerprint for a drug and finger-
prints for mutants can potentially reveal the
pathway in which the drug interacts. Hughes
et al. (28)found a significant similarity in gene
expression fingerprints between cells treated
with lovastatin, a hydroxy-methylglutaryl
CoA reductase inhibitor, and a mutant in one
of the yeast hydroxy-methylglutaryl CoA re-
ductase genes. This concept was tested further
by comparing gene expression fingerprints of
drugs with unknown targets to known mu-
tants. The fingerprint for the topical anes-
thetic dyclonine was identified as having sig-
nificant similarity to that of mutants affecting
sterol biosynthesis, and in particular, a sterol
isomerase mutant. Additional biochemical
analysis confirmed the sterol isomerase as the
likely target for dyclonine in yeast. However,
this information alone did not shed light on
the mechanism for this anesthetic in humans.
Further analysis showed that the human pro-
tein with the greatest sequence identity to the
yeast sterol isomerase was a neurosteroid in-
teractive receptor involved in regulating po-
tassium channels. If this receptor were the
target for dyclonine in humans, then a possi-
ble mechanism for dyclonine would involve
disrupting signal transduction by perturbing
potassium transport. This prediction requires
experimental verification, a requirement we
can expect from the results of most gene ex-
pression profiling studies.
Drug-target interactions have also been
explored by Marton et al. (29) using expres-
sion profiling and yeast mutants. Cells with
mutations in putative drug target proteins
were used to confirm drug interactions and
downstream effects on gene expression. The
gene expression fingerprints found in "target-
less" cells pointed to other pathway interac-
tions for the drugs tested. The ability to define
target-specific effects of drugs and their cross-
reactions with other cellular components and
pathways paves the way for future drug design
with improved targeting and specificity.
Although it is considerably more difficult to
generate specific mutations in mammalian
cells than in yeast, several technologies are
available that can allow mammalian cell sys-
tems to follow in the steps of the yeast system
for gene expression profiling and drug analysis
in mutants. The first is the production of
knock-out mutations in engineered mice. A
considerable number of knock-out mice have
already been created, and these can be ex-
plored for alterations in gene expression pro-
files in a variety of cell types (30).A more ame-
nable strategy involves the use of antisense
technology to block the expression of specific
RNAs and proteins. Antisense oligonucleo-
tides can be used as therapeutic agents as well
as tools for exploring targets (31). Cho et al.
(32) employed gene expression profiling to
identify both the specific and non-specific in-
teractions of antisense oligonucleotides. A ref-
erence library of human gene expression pro-
files for antisense oligonucleotides specific to
potential targets could conceivably be used in
the same way as the yeast mutants for identi-
fying relevant pathways and drug interactions
in the cell.
61 4 Microarrays and Gene Expression Profiling Applied to Drug Research
3.5 Molecular Toxicology
The use of microarrays, gene expression pro-
filing, and the strategies described above are
ideally suited for use in toxicology, an applica-
tion referred to as toxicogenomics. The same
gene expression data used in studying the tar-
geting of drugs also applies to toxicologic anal-
ysis of drugs where the broader effects of the
drugs are emphasized. Waring et al. (33) ap-
plied gene expression profiling to the charac-
terization of 15 known and diverse hepato-
toxins in a rat model. This study clearly dem-
onstrated that diverse drugs or toxins could
have distinct gene expression fingerprints
while sharing some similarities based on
mechanism of toxicity. Many of the genes af-
fected by each toxin encoded proteins relevant
to the known toxicity mechanisms and for pro-
teins specifically involved in the metabolism of
each toxin. These studies point to the poten-
tial use for gene expression profiling in pre-
dicting and characterizing toxic responses.
3.6 Bioinformatics Meets Chemoinformatics
The discussions above touch on only the sur-
face of the potential applications of gene ex-
pression profiling as applied to drug develop-
ment. The power of microarray technology
and gene expression profiling will likely be fur-
ther magnified by their interface with other
information-intensive tools, such as used in
chemoinformatics. For example, quantitative
structure-activity relationship (QSAR) mod-
els relate the molecular descriptors of drugs to
various biological or biochemical activities,
ranging from the inhibition activity for a par-
ticular target to more in-depth drug response
activities characterized in the NCI 60 cell
lines. As we have already discussed, measure-
ment of a single biological indicator of re-
sponse, such as cell growth inhibition, may not
reveal all the complex interactions a drug has
in the cell. However, the gene or protein ex-
pression profiles before and after exposure to a
drug hold the key to these interactions and to
true functional definitions of drug activity.
Thus, technologies that can reveal these inter-
actions promise to be the future focus for de-
signing and optimizing drugs. One approach
that might fulfill this promise would be to
combine the fields of bioinformatics with che-
moinformatics, applying QSAR modeling to
the gene expression activities affected by
drugs in a model system. Bioinformatics has
the potential to relate a group of genes af-
fected by a drug to each specific cellular com-
ponent affected by the drug, while relating
this data to the therapeutic effectiveness.
QSAR has the potential to reveal which sub-
stituents or residue substitutions in a drug an-
alog series dictate the varied interactions with
the cellular components. These data could
form the basis for designing analogs with the
highest therapeutically relevant activity and
the greatest specificity.
4 CONCLUDING REMARKS
DNA microarray technology is proving to be a
powerful genomics-based tool in a broad range
of biomedical fields. In the field of drug devel-
opment, this technology can provide insight
into the full complement of cellular targets
with which a drug might interact and assist in
defining mechanisms of action. In the context
of patient treatment, gene expression profiles
can provide information that will assist in pre-
dicting the cellular response to a drug. Fur-
ther, gene expression profiling is on the
threshold of enabling the identification of dis-
ease genes and new therapeutic targets (34).
Microarray technology is rapidly evolving, and
innovations, such as multiplexing with nanoc-
rystals embedded in microbeads (35), and fi-
ber-optic biosensor arrays (36) are being de-
veloped that will further increase the speed,
sensitivity, and power of these approaches.
Nevertheless, several engineering challenges
still exist. For example, improved detection
methods and new surface chemistries are in
demand (37).
. . , and there is a critical need for
the development and promulgation of stan-
dards in the fabrication and use of microar-
rays. There is also an enormous demand for
the develor~mentof bioinformatics tools that
will allow us to probe deeper into the vast
datasets generated by this technology to dis-
cover patterns with biological significance. Fi-
nally, many biological issues remain to be re-
solved as this technology moves into
mainstream biomedical research, such as de-
termining the appropriate normal "control"
References
tissue for comparison with disease tissue.
Thus, microarray technology is still in the
early phase of development and application in
many biomedical research fields. However, the
potential power of microarray technology is
already clear, and it is virtually certain that
the technology will play a critical role in drug
discovery and development in the near future.
5 ACKNOWLEDGMENTS
The authors gratefully acknowledge the VCU
Center for Bioelectronics, Biosensors and Bio-
chips (C3B) and the Virginia Center for Inno-
vative Technology (CITBIO-99-010) for finan-
cial support. We thank Carl Ashby and
Wanyan Xu for their assistance in the data
analysis and Jolene Windle for assistance in
writing this manuscript.
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CHAPTER TWELVE

SNPs: Single Nucleotide

~olymorphismsand
Pharmacogenomics-Individually
Designed brug Therapy
BRIAN J. PUCKETT
Virginia Commonwealth University
MCV Campus
Richmond, Virginia

STEVEN G. TERRA
JHM Health Science Center
University of Florida
Gainesville, Florida

JOE WALKER
Orchid BioSciences
Princeton, New Jersey

Contents
1 Introduction, 618
1.1 History, 618
1.1.1 Watson and Crick to the Human
Genome Project, 618
1.1.2 Drug Response and Toxicity Variabil-
ity, 618
1.2 GeneticlGenomic Definitions, 619
1.2.1 Basics, 619
1.2.2 Pharmacogenetics or Pharmacogeno-
mics?, 619
2 Single Nucleotide Polymorphisms, 620
2.1 Definition, 620
2.2 Classification of SNPs, 620
3 Technologies Used in Pharmacogenomics, 621
3.1 SNP Discovery, 621
3.2 Genotyping Technologies, 622
3.2.1 Enzymatic-Based Techniques, 622
Burger's Medicinal Chemistry and Drug Discovery 3.2.1.1 Polymerase Chain Reaction-
Sixth Edition, Volume 4: Autocoids, Diagnostics, Restriction Fragment Length
and Drugs from New Biology Polymorphism, 622
Edited by Donald J. Abraham 3.2.1.2 Single Base Primer Extension,
ISBN 0-471-37030-4 O 2003 John Wiey & Sons, Inc. 623
618 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
3.2.1.3 Pyrosequencing, 624
3.2.2 Hybridization-Based Techniques, 624
3.2.2.1 Allele-Specific Amplification, 624
3.2.2.2 DNA Array Genotyping, 624
3.2.2.3 Homogeneous Solution
Hybridization with
Fluorescence Resonance Energy
Transfer, 625
3.2.3 Combined Enzymatic/Hybridization
Techniques, 625
3.2.3.1 Invasive Cleavage Assays, 625
3.2.3.2 OLA, 625
4 Pharmacogenomics, 626
4.1 Drug Metabolizing Enzymes, 626
4.1.1 Polymorphisms in the Cytochrome
P450 System, 627
4.1.1.1 CYP2D6,627
4.1.1.2 CYP2C9, 628
4.1.1.3 CYP2C19,629
4.1.1.4 CYP3A4/5/7,629
4.1.2 Polymorphisms in Other Important
Drug Metabolizing Enzymes, 630
4.1.2.1 Dihydropyrimidine
Dehydrogenase, 630
4.1.2.2 N-Acetyltransferase-2, 630
4.1.2.3 Thiopurine Methyltransferase,
630
4.2 Polymorphisms in Drug Transporter Genes,
631
4.3 Polymorphisms in Drug Target Genes and
Clinical Efficacy, 632
4.3.1 ACE Insertiofleletion Polymor-
phism, 633
1 INTRODUCTION
1.1 History
1.1.1 Watson and Crick to the Human Ge-
nome Project. It seems to have all started at
the turn of the 20th century. Mendel's laws
came back into favor within the scientific com-
munity, leading to discoveries in the cellular
basis for heredity. But it wasn't until 1953
when Watson and Crick first described and
built the elegant model of the structure of
DNA that the field of genetics became not just
a science, but an obsession. Over the last 50
years, scientists have been feverishly trying to
unlock and decode the secrets of the human
genome. Then, a monumental accomplish-
ment was achieved when the first draft of the
entire human genome sequence was published
4.3.2 Angiotensin Type 1 Receptor, 633
4.3.3 ACE I/D and AGTRl Interaction, 635
4.3.4 5-Lipoxygenase Polymorphisms, 635
4.3.5 PI-Adrenergic Receptor
Polymorphisms, 635
4.3.6 p,-Adrenergic Receptor
Polymorphisms, 636
4.3.6.1 p,-Adrenergic Receptor SNPs,
636
4.3.6.2 p,-Adrenergic Receptor
Haplotypes, 636
4.4 Single Gene Pharmacogenetic Studies in
Genes Influencing Disease Progression, 636
4.4.1 Acute Coronary Syndromes, 636
4.4.2 Alzheimer's Disease, 638
4.4.3 Hypercholesterolemia, 639
4.5 Clinical Relevance, 641
5 Research and Development, 641
5.1 Influence of Pharmacogenomics on Clinical
Trials, 642
5.2 Use of Pharmacogenomic Data in the
Clinical Trial Process, 642
5.3 Examples from the Literature, 643
6 Obstacles Facing the Field of
Pharmacogenomics, 644
6.1 Complexity and Cost, 644
6.2 Will Pharmacogenomics Improve Medical
Care?, 644
6.3 Paradigm Shift in Health Care, 645
6.4 Ethical Considerations, 645
7 Conclusion, 646
.
in February 2001 as a result of the Human
Genome Project (1).This has sparked a re-
newed fervor for research in genomics that
will certainly carry us through and beyond the
21st century, the post-genomic era.
1.1.2 Drug Response and Toxicity Variabil-
ity. It is an incontrovertible fact that large in-
terpatient variability exists in response to
medications. Variation in response has existed
as long as medications have been used for the
prevention and treatment of disease. In many
ways, the field of pharmacogenomics began
serendipitously in the 1950s after seminal ob-
servations describing variability in response
to medications. Examples included peripheral
neuropathy from isoniazid among slow acety-
lators (2), prolonged apnea from succinylcho-
line caused by pseudocholinesterase deficiency
1 Introduction
(31,and severe hypotension from debrisoquine
among cytochrome P450 (CYP) 2D6 poor me-
tabolizers (4). For the next 40 years, pharma-
cogenetic studies focused almost exclusively
on the etiologies of altered variability in phar-
macokinetic responses to medications.
As we entered the 1990s, pharmacogeno-
mic studies began to include studies that ex-
amined pharmacodynamic variability in drug
response. Now instead of examining only dif-
ferences in drug metabolizing enzymes, scien-
tists began to focus on genes that encode drug
transporters, drug targets, and ion channels.
The goal of this chapter is to provide a primer
on pharrnacogenomics and describe how the
human genome and molecular biology tech-
niques are transforming medicine to create an
era of personalized therapeutics.
1.2 Genetic/Cenomic Definitions
1.2.1 Basics. The human genome is made
up of DNA, which is organized into 23 chromo-
somes. DNA is a double helical structure that
is composed of sugar, phosphate, and a nitrog-
enous base. The DNA strands are held to-
gether by hydrogen bonds. The four nitroge-
nous bases that make up DNA are adenine (A),
guanine (GI, cytosine (C), and thymine (T).
The base pairing is consistent in that adenine
always binds to thymine and cytosine always
binds to guanine. A combination of three base
pairs makes up a codon, and each codon spec-
ifies an amino acid that will be incorporated
into a protein. The process of making a protein
begins when an RNA polymerase attaches to a
region of DNA known as the promoter region.
This single-stranded chain now serves as a
template to synthesize a single-stranded RNA
molecule. Once RNA has been formed in the
nucleus of the cell, it is transported to the ri-
bosomes in the cytoplasm of the cell where
translation will occur. However, before trans-
lation occurs, the RNA is processed and in-
trons, non-coding regions of the DNA, are re-
moved. The removal of non-coding regions is
termed splicing. Exons, coding regions of
DNA, constitute only 5% of the human ge-
nome. Once this has occurred, the RNA mole-
cule is translated into amino acids and pro-
teins. Because there are four nucleotides, a
total of 64 different codons are possible. How-
ever, there are only 20 amino acids, hence sev-
eral different codons may specify the same
amino acid.
1.2.2 Pharmacogenetics or Pharmacogeno-
mics?. Pharmacogenetics is literally a combi-
nation of pharmacology and genetics. It thus
stands to reason that pharmacogenetics is the
study of how an individual's genetic makeup
may influence response to medications. The
field of pharrnacogenetics has been around in
one form or another for over 50 years. Histor-
ically, differences in drug response were ob-
served and documented. Based on these phe-
notypic (i.e., the outward physical effect of a
genotype) observations, scientists explored
heredity and gene lines to pharmacogeneti-
cally explain these differences in response.
Scientists further explored and concentrated
efforts on the pharmacokinetics (i.e., absorp-
tion, distribution, metabolism, and excretion)
of these medications in search of genetic dif-
ferences in, for example, how fast or slow a
particular drug is metabolized. Then came the
Human Genome Project and birth of the term
pharrnacogenomics.
There has been considerable debate about
whether the terms pharmacogenetics and
pharrnacogenomics mean the same thing or
whether they truly are different sciences.
Some state that pharmacogenomics is just the
new en vogue terminology of late. Some state
that pharmacogenetics looks at a single gene
whereas pharmacogenomics looks at multiple
interacting genes. Others state that there is a
fundamental difference in the way the re-
search problem or hypothesis is approached.
Few would argue that pharmacogenomics has
its roots in pharmacogenetics, but it is the re-
cent technology in molecular biology along
with the Human Genome Project that has
shaped and defined the field of pharmaco-
genomics. Pharmacogenetics historically re-
lied heavily on phenotypic observations to
drive hypotheses about genetic differences (5).
But, with today's technology, one can take a
genome-wide approach to apriori hypothesize
about differences in drug response and/or
search for novel drug targets. This is the es-
sence of pharmacogenomics. Furthermore,
pharrnacogenetics typically concentrated its
research in the area of pharmacokinetics thus
620 SNPs: Single Nucleotide Polyrnorphisms and Pharrnacogenornics-Individually
looking at drug-metabolizing enzyme poly-
morphism~(6). However, pharmacogenomics
opens a genomic Pandora's box, allowing the
ability to explore not only drug-metabolizing
polymorphisms, but also drug target polymor-
phism~,drug transporter polymorphisms, and
disease progression polymorphisms. If the
goal of pharmacogenetics is to explain the in-
ter-individual differences in drug response
based on genetic information, then it is the
promise of pharmacogenomics to truly indi-
vidualize pharmacotherapy (5).
2 SINGLE NUCLEOTIDE POLYMORPHJSMS
2.1 Definition
Single nucleotide polymorphisms (SNPs; pro-
nounced "snips") are single base pair substi-
tutions that occur in a sequence of DNA. It has
been estimated that SNPs occur at a fre-
quency of approximately 1 in 1000 base pairs.
Because the human genome contains approx-
imately 3 billion base pairs, there should be
approximately 30,000,000 SNPs in the ge-
nome (7). However, the number of SNPs in
coding regions (i.e., the regions that actually
code for proteins) of the genome has been es-
timated at 500,000. Recently, a "SNP map" of
the human genome was published containing
1.42 million SNPs (1). The researchers found
an average density of one SNP every 1.9 kb, or
approximately 1 in 1900 base pairs. They also
estimated that of the 1.42 million SNPs iden-
tified, only 60,000 SNPs actually fell in coding
regions. However, it is important to consider
that, although a SNP may fall outside the cod-
ing region, it may be in linkage disequilibrium
with a coding SNP, thus indirectly affecting a
biologic or pharmacologic response. Irregard-
less, in order to be classified as a polymor-
phism, it must have a frequency of at least 1%
in the population. However, to be of routine
clinical use, the frequency of polymorphisms
may need to be much higher.
2.2 Classification of SNPs
As alluded to above, there are several different
types of SNPs. Coding SNPs are those poly-
morphism~that are located within the coding
block of the gene, whereas noncoding SNPs
Designed Drug Therapy
occur outside of the coding block. Coding
SNPs can further be classified as either synon-
ymous or non-synonymous. A synonymous
SNP is a polymorphism in which, although the
codon has been changed, both the wild-type
and the polymorphic variant code for the exact
same amino acid. Thus, a non-synonymous
SNP is one in which a different amino acid is
coded and is therefore the most common SNP
described in the literature. A non-synonymous
SNP can further be classified as conservative
or non-conservative. A conservative non-syn-
onymous SNP confers an amino acid substitu-
tion of similar size and charge to that of the
original amino acid, whereas a non-conserva-
tive non-synonymous SNP substitutes an
amino acid that is very different in size and/or
charge which may greatly impact protein fold-
ing. It thus stands to reason that a non-conser-
vative nonsynonymous SNP may potentially
cause the most obvious genetic variations.
It is becoming increasingly important given
the dogma of pharmacogenomics to evaluate
multiple SNPs. A haplotype is a collection of
SNPs on a particular locus. The locus could be
multiple genes, one entire gene or merely a
segment of a gene. By examining haplotypes,
the interaction between SNPs can be further
addressed and elucidated. For example, SNPs
can often travel together because of linkage
disequilibrium, thus making it very important
to study the haplotype. It therefore may be
potentially misleading to only look at the indi-
vidual SNPs without considering their inter-
play. This has indeed been proven to be the
case in a seminal paper looking at &-adrener-
gic receptor polymorphisms is asthma (8).The
details of this case are described in more detail
in Section 4.3.6.2.
It is also very important to note that SNPs
are not the only polymorphisms out there that
affect drug response. Up until now, we have
been discussing what happens when a single
nucleotide base-pair is substituted. However,
there are also polymorphisms causing inser-
tion or deletion of a segment of DNA, splice
site mutations resulting in exon skipping, mi-
crosatellite nucleotide repeats, gene duplica-
tion, point mutations resulting in early stop
codons, and complete gene deletions. This
makes for an incredibly complex variety of
SNPs that are potentially responsible for in-
3 Technologies Used in Pharmacogenomics
ter-individual response differences observed
with certain medications.
3 TECHNOLOGIES USED IN
PHARMACOGENOMICS
The cornerstone of genomic and pharmacog-
enomic studies is the ability to accurately
identify genetic variations. The last few years
have brought the development of many low-
cost, high-throughput technologies that have
permitted investigators to address genetic
questions that were previously unapproach-
able. The molecular biology component of
pharmacogenomics research essentially in-
volves two fundamental activities: discovering
new genetic variants (i.e., SNP discovery) and
assaying for known mutations (i.e., genotyp-
ing). Selection of the most appropriate tech-
nology is based on which of these two activities
are planned. While factors such as cost, accu-
racy, and throughput requirements are al-
ways important to consider, the most appro-
priate technology for a given project is driven
by the state of knowledge concerning the gene,
locus, or disease to be studied. For example,
when there is an abundance of knowledge on
the gene sequence and variation therein, the
most appropriate technology will allow conve-
nient robust assaying of known mutations
(i.e., genotyping). When there is less informa-
tion known on gene sequence and variation
(polymorphism) within the gene, technologies
that identify new genetic variation would be
most appropriate.
3.1 SNP Discovery
A variety of techniques are available for SNP
discovery. DNA sequencing remains the most
direct method to determine the sequence of a
target gene and remains the gold standard for
detecting mutations. In this mode, an individ-
ual's sequence can be compared with many
wild-type sequences to identify new polymor-
phism~.Improvements in analytical software
and platform have made modern automated
DNA sequencers much more user friendly.
Nonetheless, the use of DNA sequencing to
identify population-wide variation is a costly,
labor-intensive endeavor. Strategies to mini-
Wild type Variant
PCR amplification
Denaturation
(formamide+ heat)
I I
Separation
(Non-denaturing
gel)
C T
Figure 12.1. Single-strand conformation polyrnor-
phism analysis. Single-stranded DNA5 are gener-
ated by denaturation of the PCR products and sep-
arated on a nondenaturing polyacrylamide gel. A
fragment with a single-base modification generally
forms a different conformer and migrates differ-
ently when compared with the wild-type DNA. Re-
produced with permission from Ref. 9.
mize the amount of sequencing allow for more
efficient use of resources.
A commonly used strategy is to use poly-
merase chain reaction (PCR) techniques.
Quite simply, one would PCR-amplify the
genes of interest and scan the PCR products
for the presence of variants. These gene scan-
ning techniques differentiate PCR products
which contain a variant sequence from those
that do not. By only sequencing positive PCR
products, gene scanning methods reduce the
amount of sequencing required to discover
new SNPs.
One of the most widely used methods for dis-
covering new variants is a conformation-based
technique called single-strand conformation
polymorphism (SSCP) analysis (Fig. 12.1).
SSCP is based on the principle that a single-
stranded DNA molecule has a specific sequence-
dependent three-dimensional structure. Se-
quence variants can be observed by running
single-stranded PCR products through a gel ma-
trix. An amplicon with a single nucleotide sub-
stitution generally forms a different conforma-
tion and will migrate differently than wild-type
DNA through a polyacrylamide gel during elec-
trophoresis. By comparing the mobility of a se-
ries of test samples with that of a control sample,
it is possible to identify those samples with a
sequence variation.
622 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
Table 12.1 Members of the SNP Consortium
AP Biotech
AstraZenecca
Aventis
Bayer AG
Bristol-Myers Squibb
Hoffman-LaRoche
GlaxoSmithKline
Novartis
Pfizer
Searle
Other popular scanning methods for SNP
discovery include chemical or enzymatic mis-
match cleavage detection, denaturing gradi-
ent gel electrophoresis, and denaturing high
performance liquid chromatography (dHPLC).
The underlying principle of these methods is
that the melting characteristics of double
stranded DNA are largely defined by its se-
quence (9).Therefore, sequence variation will
produce variable DNA denaturing and rean-
nealing, such that during electrophoresis a
single-base mismatch can produce conforma-
tional changes that result in differential mi-
gration of homoduplexes and heteroduplexes
containing these base mismatches (10).
The Human Genome Project, the SNP
Consortium, and other sequencing efforts
have produced large amounts of human se-
quence data that are available in public data-
bases. In fact, in the fourth quarter of 2001,
the SNP Consortium, which was created by
the Wellcome Trust and a group of pharma-
ceutical companies (Table 12.1),will complete
a catalog of 1,000,000 SNPs for public usage
(http://snp.cshl.org). The actual SNP identifi-
cation and mapping will be performed at the
five genomics institutes (Table 12.2). These
sequence and SNP databases will provide a
basis for rapid and efficient genome-wide SNP
discovery using data assembled from se-
quences from libraries of cDNAs. For re-
Table 12.2 Institutes Responsible f o r
Identification, Mapping, and Analysis
o f SNPs
The Sanger Centre
Whitehead Institute for Biomedical Research
Washington University School of Medicine
Stanford Human Genome Center
Cold Spring Harbor Laboratom
searchers, this represents an extremely valu-
able tool in which to do in silico searches to
identify new candidate SNPs (11).
3.2 Cenotyping Technologies
There are many options available to investiga-
tors for SNP genotyping. These vary along a
range of cost, throughput, robustness, and
convenience. In comparing currently available
and emerging technologies, a distinction
should be made between the analytical bio-
chemistries that underlie the different geno-
typing assays and the variety of platforms and
modes of detection, or readout, of the genotyp-
ing results. For instance, it is sometimes sug-
gested that DNA microarrays are a new pow-
erful method of genotyping, when in reality
DNA microarrays simply represent a means of
spatially arranging biochemical reactions,
whether hybridization alone or hybridization
linked to an enzymatic reaction, so that the
end result of the reaction can be efficiently
quantified or scored (12).
Because genotyping assays require a high
level of specificity, an additional distinction
should be made between those biochemistries
that rely primarily on differential hybridiza-
tion for their specificity and those that derive
specificity from the product of an enzymatic
reaction. By combining one of the available
allelic discrimination biochemistries with ei-
ther a solution phase or solid (array) phase
reaction platform and a common detection
methodology [electrophoresis, fluorescence,
fluorescence resonance energy transfer (FRET),
etc.1, a number of viable genotyping technolo-
gies have been developed.
3.2.1 Enzymatic-Based Techniques.
3.2.1.1 Polymerase Chain Reaction-Restric-
tion Fragment Length Polymorphism. One of
the simplest methods to genotype test samples
is through restriction fragment length poly-
morphism (RFLP)analysis. With this method,
after PCR, the PCR products are digested with
an appropriate restriction enzyme and visual-
ized by staining the gel after electrophoresis.
If the test sample contains a genetic polymor-
phism that causes a gain or loss of the restric-
tion site, then that sample will display a differ-
ent migration pattern on the gel. Appropriate
3 Technologies Used in Pharmacogenomics
Addition of extension
primer which will anneal
immediately adjacent to
(but not including) the
polymorphic site
Addition of labeled
ddNTPs and DNA
polymerase
T-* , G-•
----A
Figure 12.2. Single base primer extension. This patient is heterozygous for this A to C substitution.
The labels from both ddNTPs can be detected in this sample. In the case of a homozygous genotype,
only one of the labels would be detected. Detection might be through ELISA, fluorescence, FRET, or
FP.
quality control measures require that positive
and negative controls be present in each assay
to confirm that the restriction enzyme is ac-
tive.
The main advantages of RFLP analysis are
that it is simple to develop and use. It also does
not require expensive equipment, and it is ef-
fective for genotyping a small number of sam-
ples. The principle disadvantages are that it
is time consuming, labor intensive, and not
amenable for large numbers of samples or ge-
notypes. Another drawback is that not all
SNPs produce a usable restriction site.
3.2.1.2 Single Base Primer Extension. One
of the most effective ways to detecting poly-
morphism~is a technique known as single-
base primer extension (Fig. 12.2). In this
PCR of area that
includes polymorphic
site
Denaturation
Single stranded
PCR products
Annealing of
extension
primer
G-*
- ---- c Extension by
one base
----- ---- c ----- Detection
simple assay, a region containing the polymor-
phism of interest is first amplified in a PCR
reaction. The double-stranded PCR product is
prepared into a single-stranded DNA frag-
ment by heat or enzymatic digestion. A third
primer is then used in the primer extension
reaction. On the single-stranded PCR product,
this third primer, called the primer extension
primer, anneals immediately adjacent to, but
only including, the site of the known muta-
tion. After addition of DNA polymerase and
labeled chain-terminating dideoxynucleotides
corresponding to the wild-type and variant se-
quence, one of the two dideoxynucleotideswill
be added onto the primer at the site of the
mutation. Unlike regular deoxynucleotides,
which will extend indefinitely, dideoxynucle-
624 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
otides will only extend by one base. After com-
pletion of the primer extension reaction, de-
tection of the reaction products may occur in
any number of ways (13,14).
An important distinguishing feature of
primer extension is that assay specificity
arises from the enzymatic specificity of DNA
polymerase, not from the hybridization of the
extension primer. Consequently, single base
extension is rapidly gaining acceptance as the
reaction biochemistry of choice for high-
throughput genotyping of SNPs. It is well
suited for high-throughput applications be-
cause the reactions can occur under similar
reaction conditions, assay design and optimi-
zation are minimal, and it is portable to a va-
riety of detection platforms (15). For these
reasons, single-base primer extension is being
licensed and adapted for use of a variety of
different platforms and detection systems, in-
cluding indirect colorimetric detection on
ELISA-style microtiter plates (Orchid Bio-
Sciences' SNPStream 25K and SNPware 96
kits), DNA array product capture systems
(SNPcode kits for use on Affymetrix's Gene-
Chip and GeneFlex; Aper's APEX), fluores-
cent bead-based sorting devices (Luminex's
LabMAP), capillary DNA sequencers (ABI's
Snapshot and Amersham Pharmacia Bio-
tech's SNuPe), mass spectrometry (Seque-
nom), and fluorescence polarization (Perkin
Elmer).
3.2.1.3 Pyrosequencing. Another enzymatic
genotyping method is pyrosequencing (Pyro-
sequencing AB). In this method, primer exten-
sion is monitored by luminometric detection
of pyrophosphate, which is released on the ad-
dition of deoxynucleotides. The pyrophos-
phate is converted to ATP, which in turn stim-
ulates luciferase to produce light which can be
detected. As the deoxynucleotides are added,
the complementary DNA strand is built up
and the nucleotide sequence can be deter-
mined from the signal strength. Using pyrose-
quencing, the sequence of short 30-50 bp
stretches of DNA can be determined (15, 16).
This method available in a 96-well format and
might be useful for small low-throughput ap-
plications, but the high cost may be an issue
when high genotyping throughput is neces-
sary.
3.2.2 Hybridization-Based Techniques
3.2.2.1 Allele-Specific Amplification. The al-
lele-specific amplification (ASA)assay is based
on the finding that PCR amplification will not
occur if a primer has a mismatch at the 3' end.
Therefore, to detect the presence of a polymor-
phism, two different forward primers can be
designed for use with a common reverse
primer such that the 3' end of each forward
primer matches the expected nucleotide at the
polymorphic site for both the normal and vari-
ant sequences. A sample can be tested for the
variant by running two PCR reactions; one
with the primer set containing the normal se-
quence and the second reaction containing the
primer set containing the variant sequence.
By monitoring for the formation of allele-spe-
cific PCR products, the identity of the test
sample can be known. The results of the assay
can be detected through electrophoresis. The
addition of fluorescent dyes to primers allows
for easy multiplexing of reactions. The princi-
pal drawback of ASA based methods is the lack
of specificity because of the difficulty in opti-
mizing reaction conditions so that only per-
fectly matched oligonucleotides will be ampli-
fied.
3.2.2.2 DNA Array Genotyping. Allele-spe-
cific hybridization biochemistry underlies
many of the chip-based genotyping systems.
DNA chip-based genotyping systems allow for
the simultaneous analysis of many polymor-
phic loci on one genetic sample. Thousands or
even hundreds of thousands of allele specific
oligonucleotides are created and attached in
an order pattern onto a solid glass or silicon
surface creating an array of surface bound oli-
gonucleotide probes. Each bound oligonucleo-
tide functions as an allele-specific probe. After
PCR amplification of the areas of interest with
fluorescently labeled nucleotides, the sample
is then hybridized to the chip. Perfectly
matched probes hybridize more efficiently to
the bound oligonucleotides and therefore give
a stronger signal (10). As with other hybrid-
ization-based reactions, the drawback of this
system is the difficulty in achieving the appro-
priate stringency of hybridization conditions
so that only perfectly matched oligonucleo-
tides will be retained and provide a positive
signal.
3 Technologies Used in Pharmacogenomics
Moreover, chips must be custom made and
are therefore expensive and very inflexible.
Adding a new SNP to the analysis means that
the entire chip must be redesigned. Universal
tag array systems are being used as a reaction
sorting system and can lower the cost and in-
crease the flexibility of chip-based genotyping
systems. Many of the more specific enzyme-
based biochemistries such as single base
primer extension and oligonucleotide ligation
assay (OLA) are being developed for array-
based genotyping (17, 18).
3.2.2.3 Homogeneous Solution Hybridiza-
tion with Fluorescence Resonance Energy
Transfer. The Taqman (Applied Biosystems,
Inc.) assay uses allele-specific hybridization
to distinguish between alleles during PCR
with fluorescence resonance energy transfer
(FRET) detection. FRET detection is based on
the observation that the fluorescent emissions
from one fluorescent dye can be absorbed by
another fluorescent dye when the two are in
close physical proximity. This assay uses two
sets of probes: a pair of PCR primers and a set
of allele-specific Taqman probes. The Taqman
probes differ at the polymorphic site, such
that one probe is specific for the wild-type al-
lele, whereas the other is specific for variant
allele. Each allele-specific probe is also labeled genomic DNA are hypothetical advantages of
with both a 5' fluorescent reporter dye and a
3' quencher dye. Both probes use the same 3'
quencher dye [6-carboxy-N,N,N',N'-tetrachlo- short supply. This advantage is completely
rofluorescein (TAMRA)], whereas the wild-
type allele and variant allele probes are also
labeled with 6-carboxy-4,7,2',7'-tetrachlorofluo- (22).
rescein (TET) or 6-carboxyfluorescein (FAM),
respectively. While the probes are intact, there
is no fluorescent signal because of the close
proximity of the quencher dye to the reporter
dyes (19).
During the PCR amplification, one or both
of the allele-specific probes anneal to the poly- carries a fluorescent label. PCR is used to cre-
morphic region. During the extension phase,
the 5' reporter dye is cleaved and released by
the 5' nuclease activity of the Tag polymerase.
Cleavage releases the 5'reporter dye from the
probe, allowing it to emit its characteristic flu- polymorphic site. The 3' end of either of the
orescence (9).
Because post-PCR processing or manipula-
tion is not required, the Taqman assay is sim-
ple to perform once the assay has been opti-
mized. Unfortunately, the assay, which is
based on hybridization, requires a highly spe-
cific assay condition to deliver robust geno-
types. Also, because four oligonucleotide
probes are required (two of which must be
fluorescently labeled), these assays can be ex-
pensive to run.
3.2.3 Combined Enzymatic/Hybridization
Techniques
3.2.3.1 lnvasive Cleavage Assays. The In-
vader assay (Third Wave Technologies, Inc.) is
a FRET-based enzymatichybridization com-
bination genotyping method that offers the
potential to genotype without prior PCR am-
plification. This assay uses two hybridization
oligonucleotides (a wild-type and a variant-
specific oligonucleotide) plus an Invader
probe. The two hybridization probes partially
overlap a known polymorphic site and com-
pete for hybridization. When one probe suc-
cessfully hybridizes, it forces the other probe
into an overlapping position, which will be rec-
ognized and cleaved by the enzyme flap endo-
nuclease (20, 21). The cleaved fragment acts
as an "invader" probe in a second reaction,
where it directs the cleavage of a n end-labeled
FRET probe-template construct (9). While
avoiding PCR and genotyping directly from
the Invader assay, this requires a large .
amount of genomic DNA, which can be in
lost if investigators must perform the assay on
DNA fragments previously amplified by PCR
3.2.3.2 OLA. The OLA uses an enzymatic
reaction to increase the specificity of a hybrid-
ization-based approach. Three very specific
oligonucleotide probes are used in OLA: one
specific for the wild-type allele, one specific for
the variant allele, and a common probe that
ate amplicons containing the polymorphic
site. When the PCR products are incubated
with all three probes, the 5' region of the com-
mon probe anneals just downstream of the
allele specific probes anneals adjacent to the 5'
end of the common probe. In the presence of
thermostable DNA ligase, the two probes will
join only if there is a perfect match. The re-
sults of the assay can be observed either by gel
626 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy

Phase I Phase ll
epoxide NAT2
hydrolase

TPMT

Figure 12.3. Drug-metabolizing enzymes. Those drug-metabolizing enzyme polymorphisms that

have already been associated with altered drug effects are separated from the pie chart. For each of
the corresponding phases, the size of the pie slice approximates that drug-metabolizing enzyme's
contribution to the overall metabolism of drugs. ADH, alcohol dehydrogenase; ALDH, aldehyde
dehydrogenase; CYP, cytochrome P450; DPD, dihydropyrimidine dehydrogenase; NQO1, NADPH:
quinine oxidoreductase or DT diaphorase; COMT, catechol 0-methyltransferase; GST, glutathione
S-transferase; HMT, histamine methyltransferase; NAT, N-acetyltransferase; STs, sulfotrans-
ferases; TPMT, thiopurine methyltransferase; UGTs, uridine 5'-triphosphate glucuronosyltrans-
ferases. Reproduced with permission from Ref. 6.

electrophoresis of the ligation products or by
capture of the products on a microarray built
with a set of oligonucleotides that are comple-
mentary to a tag sequence on one of the liga-
tion probes (17). The need for three specific
probes increases the costs of this genotyping
assay.
4 PHARMACOCENOMICS
4.1 Drug Metabolizing Enzymes
More than 40 years ago, the deficiency of glu-
cose-6-phosphate dehydrogenase (GGPD) and
of the arylamine N-acetyltransferase type 2
(NAT2) were the first examples that revealed
that hereditary variants of drug-metabolizing
enzymes could be responsible for side effects
and interindividual variability in response to
drugs (23,24).Since then, significant progress
has been made in the field of pharmacogenet-
ics. Much of that work involves investigation
into the clinical relevance of genetic variahl-
ity in the enzymes responsible for the metab-
olism of both endogenous and exogenous sub-
strates (Fig. 12.3).
These drug-metabolizing enzymes, such as
the cytochrome P450s (CYP), are responsible
for the metabolic elimination of most of the
drugs currently used in medicine. Genetically
determined variability in the function of these
enzymes can have a profound effect on drug
safety and efficacy.
There are many molecular mechanisms of
variability or inactivation of drug metaboliz-
ing enzymes. These include splice site muta-
tions resulting in exon skipping (CYP2C19),
microsatellite nucleotide repeats (CYP2D6),
gene duplication (CYP2D6), point mutations
resulting in early stop codons (CYP2D61,
amino acid substitutions that alter protein
stability or catalytic activity (e.g., TPMT,
NAT2, CYP2D6, CYP2C19, and CYP2C9), or
complete gene deletions (CYP2D6).
4 Pharmacogenomics 627

Table 12.3 Selected Substrates of Polymorphic Drug Metabolizing Enzymes

Propranolol S-Warfarin Omeprazole Clarithromycin

Metoprolol Glipizide Lansoprazole Erythromycin
Timolol Glimepiride Carisoprodol Midazolam
Amitriptyline Tolbutamide Diazepam Triazolam
Nortriptyline Phenytoin Pantoprazole Cyclosporine
Imipramine Celecoxib Citalopram Tacrolimus
Desipramine Ibuprofen Clomipramine Indinavir
Paroxetine Losartan Hexobarbital Nelfinavir
Fluoxetine Irbesartan Ritonavir
Venlafaxine Torsemide Saquinavir
Codeine Cisapride
Dextromethorphan Astemizole
Tolterodine Estradiol
Propafenone Hydrocortisone
Mexiletine Progesterone
Testosterone
Sildenafil
Trazodone
Vincristine
Zaleplon
Zolpidem
Amlodipine
Diltiazem
Nifedipine
Verapamil

4.1.1 Polymorphisms in the Cytochrome Americans (26). It is involved in the metabo-

P450 System. Cytochrome P450 (GYP) en-
zymes, a very large gene family comprised of
numerous isoforms, oxidatively metabolize
xenobiotics, including many drugs. Specializ-
ing in the removal of lipophilic foreign chemi-
cals, these enzymes rank among the most
abundant proteins in the liver. Table 12.3 lists
selected medications that are metabolized by
GYP enzymes.
When CYF' mutations result in null alleles
(inactivation), a complete lack of active en-
zyme and a severely compromised ability to
metabolize drugs results. Drugs may reach
toxic plasma concentrations if given in regular
doses to these "poor metabolizers." For exam-
ple, mutations in the gene encoding cyto-
chrome P450 CYP2C9, which metabolizes
warfarin, affects patients' response to the
drug and their dose requirements (25).
4.1.1.1 CYP2D6. CYP2D6, also known as
debrisoquinelsparteine hydroxylase, is highly
polymorphic and is inactive in about 8% of
Caucasian-Americans and 2-5% of African
lism of approximately 30-40 commonly used
drugs. Millions of patients with compromised
metabolism are thus at risk of adverse drug
reactions when prescribed drugs that are
CYP2D6 substrates. Many such drugs are
used for treating psychiatric (such as antide-
pressants and antipsychotics) and cardiovas-
cular diseases (such as p-blockers and antiar-
rhythmics), where the therapeutic window
can be narrow and side effects common.
More than 70 variant alleles of the CYP2D6
locus have been described, of which at least 15
encode non-functional gene products. These
alleles as well as several functional allelic vari-
ants of CYP2D6 have been described that oc-
cur at variable frequencies in racially diverse
populations (27). Most of the null alleles have
interrupted open reading frames because of
splice-site mutations, single base deletions,
nonsense mutations, or deletion of the entire
gene. Alleles encoding non-functional full-
length proteins have also been described.
Based on genetic diagnosis, it is now possible
628 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
to identify individuals with poor metabolizer
(PM) phenotype as carriers of two null-alleles
with over 99% certainty (28-30).
Whereas using a molecular diagnostic to
identify CYP2D6 PMs has become much eas-
ier, it has remained much more difficult to pre-
dict the metabolic capacity of extensive me-
tabolizers (EM; i.e., individuals carrying one
or more functional gene copies) (30). Even in
extensive metabolizers, CYP2D6 activity is
known to vary greatly. For example, the
CYP2D6 activity represented by the metabolic
ratio (MR)values of debrisoquin and desipra-
mine have been reported to show more than a
70-fold variation among extensive metaboliz-
ers in Korean and white populations (31, 32).
In poor metabolizers, the genes often con-
tain inactivating mutations, which result in a
complete lack of active enzyme and a severely
compromised ability to metabolize drugs.
Thus, poor metabolizers of CYP2D6 are poten-
tially at risk for increased plasma concentra-
tions of drugs given at conventional doses. For
example, the metabolism of the antidepres-
sant venlafaxine is controlled by genetic poly-
morphism. Poor metabolizers of CYP2D6
have significantly reduced venlafaxine clear-
ance and an increased risk of cardiovascular
toxicity (33).
Conversely, ultrarapid metabolizers (UM)
often do not reach therapeutic concentrations
when given standard doses. In some cases,
these individuals inherit up to 13 copies of the
CYP2D6 gene, arranged in tandem (34). This
amplification polymorphism results in af-
fected people metabolizing drugs that are
CYP2D6 substrates so quickly that a thera-
peutic effect cannot be obtained at conven-
tional doses. For example, it has been esti-
mated that, whereas a daily dose of 10-20 mg
nortriptyline would be sufficient for a patient
who is a CYP2D6 poor metabolizer, an UM
inheriting multiple copies of the gene could
require as much as 500 mglday (35). Ultra-
rapid metabolizers are found in 1-10% of Cau-
casians and 2-3% of African Americans.
Among Ethiopian and Saudi Arabian popula-
tions, there is a very high frequency (2030%)
of the UM phenotype (36).
In addition to detoxifying and eliminating
drugs and metabolites, CYP2D6 is required
for activation of pro-drugs. For example, co-
deine must be converted to its active metabo-
lite, morphine, by CYP2D6, rendering the
2-8% of the population who are homozygous
for non-functional CYP2D6 alleles resistant to
the analgesic effects of this commonly used
medication. Thus, this common polymor-
phism explains at least some of the interindi-
vidual variability in pain relief from standard
doses of codeine (6).
4.1.1.2 CYP2C9. CYP2C9 is one of the
most abundant cytochromes P450 in the hu-
man liver and has been shown to metabolize
a large number of drugs, including s-warfarin,
losartan, glipizide, tetrahydrocannabinol, phe-
nytoin, torsemide, celecoxib, and various non-
steroidal anti-inflammatory drugs (37-40).
There have been six different CYP2C9 al-
leles described. The two most common variant
alleles (CYP2C9*2 and CYP2C9*3) differ from
the wild-type allele (CYP2C9*1) by a single
point mutation: CYP2C9*2 is characterized
by an Argl44Cys amino acid substitution,
whereas CYP2C9*3 has an Ile359Leu substi-
tution. CYP2C9*2 and *3 have been reported
to occur at a frequency of 8%and 6%, respec-
tively, in the Caucasian population. Both of
these variants are much less common in Afri-
can-American (1%and 0.5%, respectively) and
Asian populations (0%and 2-3%, respectively)
(41). Both allelic variants are associated with
reduced catalytic activity compared with the
wild-type. They are reported to show approxi-
mately 12% (*2) and less than 5% (*3) of wild-
type enzyme activity (42).
The potential clinical importance of these
variants was recently demonstrated. Patients
with one or more of these more common
CYP2C9 variant alleles (CYP2C9*2 or
CYP2C9*3) require a significantly lower war-
farin dose to maintain the desired level of an-
ticoagulation, and these variant alleles were
also associated with a greater likelihood of
bleeding complications (25). Patients carrying
at least one of these variants have also been
shown to require 30% less phenytoin to
achieve therapeutic phenytoin concentrations
(42).
Much less is known about the less common
CYP2C9 variants. CYP2C9*4 results in an
Ile359Thr substitution. It has been reported
to be extremely rare (43). CYP2C9*5 is re-
ported to lead to an Asp360Glu substitution.
4 Pharmacogenomics
The CYP2C9*5 variant has only been ob-
served in African Americans, such that ap-
proximately 3% of this population carries the
CYP2C9*5 allele. In vitro intrinsic clearances
for CYP2C9*5, calculated as the ratio of V ,/
K,, ranged from 8 to 18%of CYPZCS*l values
in the initial report (44). The CYP2C9*6 vari-
ant results in a frameshift mutation. To date,
it has only been observed in one Caucasian
patient (R. S. Kidd, personal communication).
4.1.1.3 CYP2C19. CYP2C19, also known
as mephenytoin hydroxylase, was first de-
scribed in 1993 (45). Since then, eight alleles
have been identified. Each of the alleles other
than CYP2C19*1 have been associated with
almost complete absence of gene expression.
Most of the alleles (CYP2C19*2, *3, *4, *5, "6,
*7, and *8) occur infrequently (approximately
3-5% in total) in random Caucasian and Afri-
can-American populations. In all racial groups
studied, CYP2C19*2 is the allele most com-
monly associated with an inactive gene prod-
uct. Within Asian populations, the higher fre-
quency of CYP2C19*2 and nd2C19*3 alleles
accounts for the higher prevalence in this ra-
cial group (approximately 35% PM) (46,471.
CYP2C19 metabolizes many clinically im-
portant drugs (Table 12.3). Subjects with the
CYP2C19 PM phenotype have an area under
the curve (AUC) of omeprazole that is more
than sixfold higher than efficient metabolizers
(EM), and the drug has a severely prolonged
half-life in PM individuals (48). A similar rela-
tionship is seen for other proton pump inhibi-
tors (49). To reach similar plasma levels, PMs
of CYP2C19 would take about 1-2 mg of ome-
prazole instead of the recommended dose of 20
mg (50).
Regarding proton pump inhibitors, the ef-
fect of CYP2C19 PM status is not limited to
pharmacokinetic alterations. The difference
in the pharmacokinetics has been shown to
influence the outcome of H. Pylori eradication
therapy. Furuta et al. showed that in patients
with confirmed H. Pylori infection treated
with omeprazole or lansoprazole plus cla-
rithromycin and amoxicillin, CYP2C19 PMs
had an eradication rate of 97.8% compared
with a rate of 72.7% (P < 0.001) for CYP2C19
EMS (51).
In addition to the proton pump inhibitors,
CYP2C19 genotype has also been shown to be
associated with reduced elimination of diaze-
pam (52), proguanil (53), imipramine (54),
citaloprarn (55), carisoprodol (56), and hexo-
barbital (57).
4.1.1.4 CYP3A4/5/7. The CYP3A family
consists of CYP3A4, CYP3A5, and CYP3A7.
The CYP3A members are the most abundant
CYPs in the human liver and small intestine.
Substantial interindividual differences in
CYP3A expression, exceeding 30-fold in some
populations, contribute greatly to variation in
oral bioavailability and systemic clearance of
CYP3A substrates (58). One factor contribut-
ing to this large variability in 3A expression is
the presence or absence of CYP3A5. CYP3A5
was previously detected in livers and small in-
testines of only some adult individuals, but the
basis for this "polymorphic" expression was
unknown (59,60).
Recently, two important SNPs in CYP3A5
(CYP3A5*3 and *6) were described. Relative
to the wild-type (CYP3A5*1),these mutations
have been shown to cause alternative splicing
and protein truncation, which results in the
absence of CYP3A5. Only carriers of at least
one CYP3A5*1 allele have been shown to ex-
press large amounts of CYP3A5. The ethnic
distribution of the CYP3A5*1 allele indicates
that relatively high levels of CYP3A5 are ex-
pressed by an estimated 30% of Caucasians, .
30%of Japanese, 30%of Mexicans, 40% of Chi-
nese, and more than 50% of African Ameri-
cans, Southeast Asians, Pacific Islanders, and
Southwestern American Indians (58).
For most Caucasians and African Ameri-
cans who carry the CYP3A5*1 allele, CYP3A5
accounts for at least 50% of the total CYP3A
content. Because most CYP3A4 substrates are
also substrates for CYP3A5, this CYP3A5
polymorphism influences overall CYP3A ac-
tivity in humans (61). Thus, the presence or
absence of CYP3A5 should contribute sub-
stantially to the total metabolic clearance of
the many CYP3A substrates. Indeed, those
heterozygous or homozygous for CYP3A5*1
should have the highest clearance and lowest
oral bioavailability of CYP3A substrates.
Moreover, these people might be more likely to
encounter a lack of efficacy from standard
doses (58).
Important variation in other clinically rel-
evant CYP enzymes such as CYPlA2,
630 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
CYP2A6, and CYP2E1 has been demonstrated
and reviewed in detail elsewhere (50).
4.1.2 Polymorphisms in Other Important
Drug Metabolizing Enzymes
4.1.2.1 Dihydropyrimidine Dehydrogenase.
Dihydropyrimidine dehydrogenase (DPD) is
the initial and rate-limiting enzyme in the ca-
tabolism of the chemotherapeutic agent 5-flu-
orouracil (5-FU). Diasio et al. conducted a fa-
milial study that suggested an autosomal
recessive pattern of inheritance of deficiency
of DPD. While it seems that a complete ab-
sence of DPD activity is extremely rare, even
partial enzyme inactivity might result in se-
vere toxicity from 5-FU (62). Prospectively
evaluating the gene encoding DPD is a typical
example of what could be a useful pharmaco-
genomic approach to preventing toxicity from
a very effective drug that has a high level of
toxicity (63).
4.1.2.2 N-Acetyltransferase-2. The N-acetyl-
transferase-2 (NAT2) polymorphism is one of
the most common polymorphisms known in
human populations. While more than 50% of
Caucasians are NAT2 slow acetylator pheno-
type, there is a tremendous amount of inter-
ethnic variation in the frequency of the slow
acetylator polymorphism (64). For instance,
the slow acetylator phenotype is much more
frequent in Egyptians but is much less fre-
quent in Asians (65).
This polymorphism (NAT2)was discovered
almost 50 years ago after differences were ob-
served to isoniazid toxicity in tuberculosis pa-
tients (66). Subsequently, the differences in
isoniazid toxicity were attributed to genetic
variability in NAT2, a cytosolic phase I1 con-
jugation enzyme primarily responsible for
deactivation of isoniazid (67). Indeed, the poly-
morphism was termed the "isoniazid acetyla-
tion polymorphism" for many years until the
importance of the polymorphism in the metab-
olism and disposition of other drugs and chem-
ical carcinogens was fully appreciated (65).
Since these first observations, a wealth of
clinical evidence has shown that the disposi-
tion of a variety of drugs (including sulfon-
amides, dapsone, hydralazine, procainarnide,
and caffeine) possessing primary aromatic
amino or hydrazine functional groups is af-
fected by the same genetic defect (68). In ad-
dition to metabolizing drugs, NAT2 is also
known to catalyze both N-acetylation (usually
deactivation) and 0-acetylation (usually acti-
vation) of aromatic and heterocyclic m i n e
carcinogens. Epidemiological studies suggest
that the NAT2 acetylation polymorphisms
modify risk of developing urinary bladder,
colorectal, breast, head and neck, lung, and
possibly prostate cancers. Associations be-
tween slow NAT2 acetylator genotypes and
urinary bladder cancer and between rapid
NAT2 acetylator genotypes and colorectal
cancer are the most consistently reported (65).
The importance of the NAT2 polymorphisms
in clinical pharmacology and toxicology has
been extensively reviewed (68,69).
Grant et al. first demonstrated that the
classical isoniazid slow acetylator phenotype
is due, at least in part, to reduction of the ex-
pression of NAT2 protein (70). No polymor-
phism~in the 5' or 3' regions of the gene have
been reported. Eleven SNPs have been identi-
fied in the NAT2 coding region. Five of these
are capable of producing the slow acetylator
phenotype. The four most common of these
are NAT2*5, NAT2*6, NAT2*7, and NAT2*14.
NAT2*4 is associated with the rapid acety-
lator phenotype and is considered the wild-
type allele because of its absence of any of
these substitutions. However, NAT2*4 is
not the most common allele in many ethnic
groups, including Caucasians and Africans
(71, 68).
4.1.2.3 Thiopurine Methyltransferase. Aza-
thioprine, thioguanine, and 6-mercaptopurine
are thiopurine drugs that are used to treat
acute lymphoblastic leukemia, autoimmune
disorders, inflammatory bowel disease, and
organ transplant recipients. These drugs are
metabolized by the genetically polymorphic
enzyme thiopurine methyltransferase (TPMT).
Thiopurines are very useful drugs, but they
have a relatively narrow therapeutic index,
with life-threatening myelosuppression as a
major toxicity (72).
Population studies have found that approx-
imately 11%of Caucasians are heterozygous
and 0.3% homozygous for TPMT deficiency
(73). For the TPMT polymorphism, all pa-
tients who inherit two non-functional TPMT
alleles will develop dose-limiting hematopoi-
4 Pharmacogenomics

Table 12.4 Selected Substrates, Inducers, and Inhibitors of P-

Glycoprotein (138)
P-Glycoprotein P-Glycoprotein P-Glycoprotein
Substrates Inhibitors Inducers
Amiodarone Amiodarone Dexarnethasone
Anthracyclines Atorvastatin Phenobarbital
Cisplatin Clarithromycin Phenytoin
Cyclosporine Cyclosporine Rifampin
Cytarabine Diltiazem St. John's Wort
Dactinomycin Erythromycin
Daunorubicin Itraconazole
Dexamethasone Ketoconazole
Digoxin Quinidine
Docetaxel Quinine
Doxorubicin Ritonavir
Etoposide Tacrolimus
Fexofenadine Tamoxifen
Fluorouracil Verapamil
Glucocorticoids
Indinavir
Loperamide
Losartan
Methotrexate
Mitoxantrone
Nelfinavir
Paclitaxel
Ritonavir
Saquinavir
Sirolimus
Tacrolimus
Topotecan
Vinblastine
Vincristine
Vindesine
Vinorelbine

etic toxicity. Patients deficient in this drug-
metabolizing enzyme can require up to a 15-
fold reduction in mercaptopurine to prevent
fatal hematotoxicity (74-77).
The full clinical and molecular implications
of this variant have recently been reviewed
(72, 78).
4.2 Polymorphisms in Drug Transporter
Genes
P-glycoprotein (P-gp) is an ATP-dependent
drug efflux pump. In humans, P-gp is encoded
by the multi-drug resistance gene (MDR-1)
that is located on the long arm of chromosome
7. Overexpression of P-gp in neoplastic cells is
associated with the phenomenon of multi-
drug resistance to chemotherapeutic agents
by promoting efflux of chemotherapy (79). P-
glycoprotein is also expressed in normal cells
including the intestinal epithelium, renal
proximal tubule, liver, adrenal cortex, pla-
centa, testes, and blood-brain barrier. Re-
cently, P-gp has been implicated as the caus-
ative factor in numerous pharmacokinetic
interactions (80). For example, amiodarone
and quinidine therapy increases serum
digoxin concentrations through inhibition of
P-gp in the intestine and renal tubule, which
increases digoxin absorption and decreases to-
tal body clearance. Table 12.4 provides a par-
tial list of substrates, inhibitors, or inducers of
P-gp. Considerable substrate overlap and tis-
sue location exist between P-gp and the CYP
3A4 isoenzyme.
632 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
Table 12.5 Selected Polymorphisms Associated With Altered Drug Response
GeneIGene Product Medication
&OX5 Lipoxygenase inhibitors
Angiotensin 1 receptor Losartan
PI-Adrenergicreceptor Metoprolol
@,-Adrenergicreceptor Albuterol
Bradykinin B2 receptor ACE inhibitors
Dopamine D2 receptor Dopamine antagonists
Estrogen receptor Equine estrogen
Gsa p-blockers
Platelet Fc Heparin
Serotonin transporter SSRIs
Sixteen SNPs have been identified to date
in the MDR-1 gene (81). Most of the polymor-
phisms are either synonymous or occur in in-
tronic regions. However, one synonymous
polymorphism in exon 26 (C3435T) has func-
tional significance by influencing the expres-
sion of P-gp in the intestine. Individuals with
the TT genotype have a twofold lower expres-
sion of P-gp compared with the CC genotype
(82). Thus, one could predict that individuals
with the TT genotype would have higher con-
centrations of drugs that are P-gp substrates
and indeed this is the case. In one study, sub-
jects with the TT genotype had a statistically
significant 38% higher digoxin C, concen-
tration compared with subjects with the CC
genotype (82). Therefore, patients with the
TT genotype would also be expected to have
higher conceintrations of other P-gp substrates.
Alternatively, CC homozygotes through in-
creased expression of P-gp may experience
subtherapeutic concentrations of P-gp sub-
strates and experience therapeutic failures.
Genotype frequencies at exon 26 are highly
dependent on the ethnicity of the population
studied. One recent investigation examined
1280 subjects from 10 different ethnic groups
(British Caucasians, Ghanaians, Kenyans, Af-
rican Americans, Sudanese, Portuguese,
Southwest Asians, Chinese, Filipinos, and
Saudis) (83). The frequency of the T allele (as-
sociated with lower expression of P-gp) was
significantly lower in the African populations
compared with the Asian and Caucasian
groups (16-27% versus 41-66%). Based on
this finding, persons of African descent would
be expected to have higher P-gp expression
Effect Associated with Polymorphism Reference
Improvement in FEV,
Reduction in mean arterial pressure
Reduction in blood pressure
Improvement in FEV,
ACE inhibitor induced cough
Anxiolytic/antidepressiveeffects of
neuroleptics
Increased bone mineral density
Reduction in blood pressure
Heparin induced thrombocytopenia
Antidepressant response 144,145
compared with Caucasians and Asians. Conse-
quently, plasma concentrations of drugs that
are P-gp substrates may be lower among Afri-
can populations than those of other racial
backgrounds despite equivalent doses. Sup-
porting this is a study that revealed that Afri-
can-American subjects had lower plasma cy-
closporine concentrations compared with
Caucasians given equivalent doses (84). Afi-i-
can-American patients have a higher rejection
rate after transplantation compared with
Caucasians. Plausibly, the higher frequency of
the C allele among African Americans may re-
sult in subtherapeutic concentrations of P-gp
substrates tacrolimus and cyclosporine and
therefore increased risk of rejection. Likewis'e,
the HIV-protease inhibitors may be less effec-
tive among patients with the CC genotype and
may have diminished penetration into the
central nervous system. Clearly, further study
is needed to determine the association be-
tween the C3435T polymorphism with treat-
ment outcomes among patients receiving P-gp
substrates.
4.3 Polymorphisms in Drug Target Genes
and Clinical Efficacy
The biomedical literature contains a multi-
tude of publications that attempt to correlate
genetic variation underlying differential re-
sponse to medications. It is obviously not fea-
sible to review all of the examples from the
literature, however, Table 12.5 summarizes
several of these polymorphisms that are asso-
ciated with altered drug response. Our goal is
to provide several illustrative examples of how
4 Pharmacogenomics
polymorphisms in drug targets are being used
to establish a personalized medicine platform.
4.3.1 ACE Insertion/Deletion Polymor-
phism. One of the most studied polymor-
hhisms occurs in the gene encoding angioten-
sin-converting enzyme. This polymorphism
occurring in intron 16 is not a SNP. Rather it
is an insertionldeletion (ID) of a 287-base pair
product. Numerous studies have demon-
strated that the D allele is associated with
higher concentrations of the hormone, angio-
tensin I1 (85). The functional consequences of
these findings are apparent and have been
supported by several examples from the liter-
ature demonstrating that the D allele is asso-
ciated with increased risk of hypertension,
myocardial infarction, and ventricular ar-
rhythmias (86-88). Presence of the D allele is
also associated with a poorer prognosis among
patients with heart failure (89). Among 328
-
~ a t i e n t with
s heart failure followed at a car-
diomyopathy clinic, after 2 years, the percent-
age of patients with transplant-free survival
was 78% for the I1 genotype, 65% ID, and 60%
DD (P= 0.044). Interestingly, these investiga-
tors also examined the impact of the ACE I/D
polymorphism with P-blocker therapy in this
cohort of patients. In the 208 patients who
were not receiving P-blocker therapy, the
2-year transplant-free survival was 81% 11,
61%ID, and 48% DD. However, a provocative
finding was that P-blocker therapy obviated
the influence of the D allele with poor progno-
sis. Among patients receiving p-blockers, the
2-year transplant-free survival was 70% for I1
genotype, 71% for ID, and 77% DD (P = NS).
Table 12.6 reviews several studies examin-
ing the impact of the insertioddeletion poly-
morphism with clinical response to ACE in-
hibitor therapy. Review of these studies
reveals numerous conflicting results. Several
possibilities exist for these equivocal findings
including heterogeneous patient populations,
sample size, endpoints, and study duration.
However, perhaps the largest factor resides in
the limitation of examining a single SNP.
Medications act with numerous transporters
and receptors to illicit a therapeutic response.
Consequently, it is more plausible that vari-
ability in drug efficacy will be caused by poly-
morphism~in multiple genes involved in the
drug response pathway. Thus, the findings
with the ACE I/D polymorphism provide a ra-
tionale for future studies to eschew the sim-
plicity of examining a single SNP and instead
incorporate a more genomic approach.
One such effort examined 45 polymor-
p h i s m ~in genes encoding angiotensinogen,
ACE, and the AT1 receptor (90). A total of 91
patients who had received an ACE inhibitor
for the treatment of hypertension were retro-
spectively studied, and 10 polymorphisms as-
sociated with a good response were identified.
These 10 polymorphisms, termed "genetic sig-
natures," were then prospectively evaluated
in 102 patients with hypertension who re-
ceived ACE inhibitors. The response rate to
ACE inhibitor therapy was 73%among the pa-
tients with the "genetic signatures" compared
with 42% response rate in the cohort without
the "genetic signatures." Thus, while focusing
on a single SNP is easy, it is unlikely to yield
sufficient predictive accuracy to be useful clin-
ically. Future pharrnacogenomic studies will
need to examine the impact of several SNPs
and haplotypes to correlate these "genetic sig-
natures" with drug variability.
4.3.2 Angiotensin Type 1 Receptor. Angio-
tensin I1 produces its deleterious effects by in-
teractingwith the angiotensin type 1receptor.
A well-described polymorphism exists in the
gene encoding the angiotensin type 1 receptor
with either an adenine (A) or cytosine (C) at
position 1166 of the 3' untranslated region of
the gene. Case-control studies have shown an
association between the C allele and increased
risk of hypertension, aortic stiffness, and a
worse prognosis among patients with idio-
pathic heart failure (91-93). Thus, angioten-
sin receptor blockers used in the management
of hypertension and heart failure may be par-
ticularly effective among patients with the C
allele. One study of healthy volunteers demon-
strated that subjects carrying at least one C
allele experienced a larger decrease in mean
arterial pressure and an increase in glomeru-
lar filtration rate compared with subjects with
the AA genotype (94). Although this finding
warrants further study, it suggests that the C
allele may be an important predictor of re-
sponse to angiotensin receptor blockers. Be-
cause this polymorphism occurs in an un-
 Table 12.6 Impact of Angiotensin-ConvertingEnzyme Polymorphisms and Response to ACE Inhibitor Therapy
Study Population Results
27 Healthy volunteers Captopril significantly reduced mean arterial pressure in the I1 and ID genotypes; no significant
change in the DD group. Renal vascular resistance was significantly reduced in the I1 and ID
genotypes with no significant change in the DD group (148).
27 Healthy volunteers No correlation between ACE genotype and reduction in mean arterial pressure after single dose of
enalapril(149).
530 Patients with diabetes Largest reduction in albumin excretion rate occurred in lisinopril treated patients with the I1
c
.p genotype (150).
212 Non-diabetic patients with proteinuria Largest reduction in proteinuria occurred in ramipril treated patients with the DD genotype
(151).
104 Patients with hypertension Statistically significantly greater reductions in systolic and diastolic blood pressure after 6 months
of fosinopril therapy among patients with the DD genotype compared with I1 or ID genotypes
(152).
57 Patients with hypertension Absolute and percent reduction in diastolic blood pressure tended to decline more after 6 weeks of
imidapril therapy among hypertensive patients with the 11genotype compared with ID and DD
genotypes (153).
4 Pharrnacogenomics
translated region of the gene, it is likely that
this polymorphism is in linkage with another
marker.
4.3.3 ACE I/D and AGTRI Interaction. In
routine clinical practice, risk factors for dis-
ease tend to be either additive or synergistic.
In general, a patient with hypertension, diabe-
tes, and hyperlipidemia is at greater risk of
suffering an acute coronary event compared
with a patient with only one of these risk fac-
tors. Thus, interactions among polymor-
phism~may also be germane to pharmaco-
genomic studies. To this end, several studies
have examined the impact of both the ACE DD
and AGTRl CC genotype. One study demon-
strated an increased risk of myocardial infarc-
tion among patients with the DD and CC ge-
notypes for the ACE and AGTRl genes,
respectively (95). However, a recent case-con-
trol study failed to demonstrate an association
between these two polyrnorphisms with the
risk of myocardial infarction (96). Another
study revealed that the risk of ventricular ar-
rhythmias among patients with systolic heart
failure was significantly increased in patients
with the DD ACE genotype and the CC geno-
type for the AT1 gene (97). Thus, once again,
these results suggest that this group of pa-
tients may derive particular benefit from more
aggressive management of their disease.
4.3.4 5-Lipoxygenase Polymorphisms. Leuko-
trienes mediate airway inflammation and play an
integral role in the pathophysiology of asthma.
Zileuton (Zyflo) inhibits the enzyme 5lipoxygn-
ase, reducing the formation of leukotrienes and
thus improves the symptoms of asthma. A poly-
morphism exists in the gene encoding the 5-lipoxy-
genase (ALOX5) promoter region. This polymor-
phism contains three to six tandem repeats of
GGGCGG. A recent study examined the impact of
the tandem repeat polymorphism in the L O X 5
promoter region with response to an investiga-
tional 5-lipoxygnase inhibitor (98). The clinical
outcome in the study was percentchange in forced
expiratory volume in 1 second (FEV,)from base-
line. The allele frequency for the wild-type L O X 5
promoter polymorphism was 0.77. Patients who
were homozygous or heterozygous for the wild-
type allele had an average change in FEV, from
baseline of 18% and 23%, respectively. Patients
with no wild-type alleles had a 1%decrease in
FEV, from active treatment.
4.3.5 &-Adrenergic Receptor Polymor-
phism~.The pl-adrenergic receptor (AR) is a
G-protein-coupled receptor expressed in a
number of cell types including the heart and
kidneys. The gene that codes for the pl-AR is
intronless and is located on chromosome
10q21. There are two common SNPs within
the PI-AR gene at codon 49 and codon 389
(99).
Codon 49 is located in the extracellular tail
of the amino terminus end of the receptor, a
potentially important region for receptor
binding, regulation, and expression (100). A
non-synonymous SNP produces a glycine
(Gly) for serine (Ser) substitution at codon 49
(Ser49Gly).Although there are no data in viuo
associating this polymorphism with drug re-
sponse, a recent site-directed in uitro mu-
tagenesis study suggests that agonist-pro-
moted down-regulation of the receptor is
amplified with the Gly49 variant (101).
Codon 389 is located in the intracellular tail
of the carboxy terminus end of the receptor, a
potentially important region for G-protein
coupling (100). A non-synonymous SNP pro- ,
duces a Gly for arginine (Arg) substitution at
codon 389 (Arg389Gly). This polymorphism
has been shown to vary by race, with African
Americans possessing an allele frequency of
42% for the Gly389 variant while Caucasians
possess a frequency of only 27% (102). Fur-
thermore, in uitro mutagenesis studies have
shown a functional difference with this poly-
morphism (103). In this study, those cells car-
rying the Arg389 variant had a nearly twofold
greater resting activity rate, as measured by
adenylyl cyclase levels, and an almost fourfold
greater activity when stimulated with p-ago-
nist, thus suggesting the Gly389 is a less active
or perhaps less reactive receptor form. This
theory has been recently put to the test in a
prospective study of patients with hyperten-
sion, and indeed, this polymorphism may be
an important determinant of the antihyper-
tensive response to p-blocker therapy (104).
Further studies are needed to evaluate
whether this polymorphism confers the racial
636 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
differences observed in response to p-blocker
therapy in both hypertensive and heart failure
patients.
4.3.6 p,-Adrenergic Receptor Polymorphisms
4.3.6.1 P,-Adrenergic Receptor SNPs. The
P2-adrenergicreceptor is a G-protein-coupled
receptor that interacts with endogenous cat-
echolamines and various pharmacologic
agents. The mainstay of therapy for acute
bronchoconstriction is the administration of
p,-AFt agonists such as albuterol. A non-syn-
onymous SNP in the P2-AR gene produces
a Gly for Arg substitution at codon 16
(Argl6Gly). In vitro studies indicate that the
Gly16 form of the receptor undergoes en-
hanced agonist-mediated downregulation
compared with the Arg allele (105). These
findings have been supported by several clini-
cal studies. One study demonstrated that sub-
jects with the Argl6 homozygous genotype
were 5.3 times more likely to have a positive
bronchodilator response to a single dose of al-
buterol compared with Gly16 homozygotes
(106). Another study enrolled 16 patients with
asthma and measured FEV, response to an 8
mg oral dose of albuterol(107). The study pop-
ulation was divided into two groups: Argl6 ho-
mozygotes and Gly16 homozygotes. Patients
who were homozygous for Argl6 had a four-
fold greater FEV, response cornbared with
Gly16 homozygotes despite nearly identical
plasma albuterol concentrations. Moreover,
the codon 16 genotype may also influence
long-term response to albuterol. One study of
107 patients with mild-to-moderate asthma
demonstrated that Argl6 homozygous pa-
tients receiving regularly scheduled albuterol
had nearly double the number of asthma exac-
erbations per year compared with placebo.
Furthermore, the rate of asthma exacerba-
tions was significantly greater among k g 1 6
homozygotes during treatment with albuterol
compared with heterozygotes and Gly16 ho-
mozygotes (108). A separate study found that
k g 1 6 homozygotes receiving regularly sched-
uled albuterol experienced a decrease in morn-
ing peak expiratory flow rate (PEFR). In con-
trast, Gly16 homozygous patients who
received regularly scheduled albuterol did not
experience a decline in PEFR (109). These re-
sults suggest that the Argl6 homozygous ge-
notype is associated with deleterious effects
from regularly scheduled albuterol therapy
and that patients with this genotype should
only receive albuterol for breakthrough symp-
toms.
4.3.6.2 p,-Adrenergic Receptor Haplotypes.
In all, 13 polymorphisms occur in the P,-AR
gene. Thus, if these polymorphisms occurred
completely randomly, one would expect a total
of 213 variations (haplotypes) in the P,-AR
gene. However, only 12 haplotypes occur, and
5 haplotypes describe 88% of the population
(8). Thus, the polymorphisms in the p,-AR are
in strong linkage disequilibrium. A seminal
paper has investigated the impact of haplo-
types, rather than individual SNPs in predict-
ing response to albuterol among patients with
asthma. Importantly, there was no association
between an individual SNP and remonse to
albuterol. However, haplotype pair was signif-
icantly related to improvements in FEV, from
albuterol (8). Therefore, examination of mul-
tiple SNPs in a receptor that is physiologically
linked to drug response resulted in the best
prediction of therapeutic efficacy. Focusing on
multiple genes and/or multiple SNPs to deter-
mine disease associations or drug response is
analogous to the multiple factors that a clini-
cian must consider when dosing a medication.
For example, when prescribing digoxin, the
likelihood of prescribing the appropriate dose'
is increased when a clinician considers multi-
ple factors such as patient age, body size and
weight, renal function, and concomitant drug
therapy.
4.4 Single Gene Pharmacogenetic Studies in
Genes influencing Disease Progression
Once again, it would not be feasible to review
all the examples in the biomedical literature of
polymorphisms influencing disease severity or
progression. However, Table 12.7 summarizes
many of these polymorphisms that influence
disease severity and associated drug response.
We will discuss a few of these disease states in
detail.
4.4.1 Acute Coronary Syndromes. Several
studies have examined the impact of SNPs on
the natural history of patients undergoing
percutaneous coronary intervention (PCI) for
the treatment of acute coronary syndromes.
 Table 12.7 Selected Polymorphisms Muencing Disease Severity and Associated Drug Response
Gene Altered Disease Severity Impact of Polymorphism on Drug Response Reference
ACE D allele f risk of death or need for heart P-Blockers abolished poorer prognosis of patients with
transplant DD genotype
APOE E4 allele f in Alzheimer's disease Presence of E4 allele! associated with poor response to
tacrine
APOE Smokers with an E4 allele had a threefold Unknown
f risk of CHD event
P1 Heart failure patients with the Ser49Ser Unknown
genotype had a worse 5-year prognosis
compared with patients with a Gly
variant
Reduced survival rate among heart failure Unknown
patients with an Ile164 allele
Reduced exercise capacity among heart Unknown
failure patients with an Ile164 allele
Calcitonin Association between heterozogosity and Unknown
fracture risk among postmenopausal
QI
W women
V
Cystathionine beta Risk of coronary artery disease Response to homocysteine lowering from folic acid
synthase
Endothelin A T frequency of TT homozygotes in Unknown
idiopathic dilated cardiomyopathy
Factor V Risk of venous thrombosis TRisk of venous thrombosis from oral contraceptives
Factor VII Patients with the ArgArg genotype have a Unknown
threefold f risk of complications after
PC1
Odds ratio of MI among patients with Unknown
ArgGln genotype = 0.47 (0.27-0.81)
compared with ArgArg genotype
GP IIIa Association between PlA2allele and acute Unknown
coronary thrombosis
G-protein Increased BMI among TT homozygous Unknown
primiparous women
HERG, Long QT syndrome .T Risk of drug induced Torsade de Pointes
KvLQTl,
MiRPl
P-selectin Risk of myocardial infarction Unknown
Prothrombin Risk of venous thrombosis .T Risk of venous thrombosis with oral contraceptives
CHD, coronary heart disease; PCI, percutaneous coronary intervention; MI, myocardial infarction; GP, glycoprotein; BMI, body mass index.
638 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
One of the major complications from PC1 is
restenosis of the target coronary artery, which
occurs in as many as 40% of patients. Resteno-
sis results in substantial morbidity and in-
creases need for repeat coronary interven-
tions. One study examined a polymorphism in
the gene that encodes Factor VII. Factor VII
has an integral role in the process of clot for-
mation among patients with acute coronary
syndromes. After plaque rupture, tissue factor
released from the lipid core complexes with
Factor VII, leading to the activation of Factor
X, and ultimately Factor IIa (thrombin). This
group of investigators studied 666 consecutive
patients undergoing PC1 (110). A polymor-
phism (Arg353Gln)in exon 8 of the Factor VII
gene was studied to see if it influenced the rate
of death, myocardial infarction, or urgent tar-
get vessel revascularization within 30 days of
PCI. An earlier study showed the Gln allele
was associated with a 20-30% lower Factor
VII concentration (111).Thus, one would ex-
pect the Gln allele to be associated with re-
duced cardiovascular complications. Indeed,
the incidence rate for the composite endpoint
was 7.7% among Arg353Arg homozygotes
compared with 2.5% among patients carrying
at least one Gln allele (RR of 0.32; 95% CI,
0.08-0.90). Although statistically significant,
the results are limited by the small number of
events (43) in this study. Ironically, Factor VII
concentrations were similar between Arg ho-
mozygotes and Gln carriers who reached the
primary endpoint. Thus, the putative mecha-
nism for this reduced risk among Gln carriers
remains to be elucidated.
A second study examined a polymorphism
in the glycoprotein IIIa gene. This protein
plays an integral role in the final common
pathway of platelet activation and fibrinogen
cross linkage, resulting in the formation of a
platelet plug. The two common allelic isoforms
are PIA1and PlA2.A retrospective study dem-
onstrated an association between the PlA2
polymorphism with a heightened risk of acute
coronary thrombosis (112). These findings
were confirmed in a prospective study that ex-
amined the association between the GP IIIa
PIA1and PlA2isoforms with angiographically
confirmed restenosis. The study population
consisted of 1150 patients undergoing PC1
with stent placement who also had a follow-up
cardiac catheterization at 6 months (113). Ap-
proximately 72% of the study population were
PIA1 homozygotes, 25% were heterozygotes,
and 3% were PlA2homozygotes. At 6 months,
47% of patients with the PlA2allele had angio-
graphically confirmed restenosis compared
with 38% for PIA1homozygotes (OR 1.42; 95%
CI, 1.09-1.84). The association between geno-
type and restenosis was strongest among PlA2
homozygotes and women.
Although both studies demonstrate a sta-
tistically significant association between the
studied polymorphism and complications af-
ter PCI, the results need to interpreted with
consideration of the absolute risk reduction
(ARR) and number needed to treat (NNT). A
corollary to NNT, is the number needed for
genetic effect (NNGE). The NNGE is calcu-
lated as 11ARR. Thus, when examining the im-
pact of the Arg353Arg genotype on increased
complications after PCI, only 1 of 19 patients
with this genotype would experience a compli-
cation after PC1 (114). Likewise, in the study
of the PlA2 polymorphism, only 1 of 11 pa-
tients with the PlA2 allele will have angio-
graphically confirmed restenosis.
4.4.2 Alzheimer's Disease. One of the most
well-studied disease association genes is the
apolipoprotein E (APOE) gene located on
chromosome 19; this is the gene related with
the risk of developing Alzheimer's disease.
Three allelic variants exist for APOE. The fre-
quency for E3, E4, and E2 are 77%, 15%,and
8%, respectively. Several studies have deter-
mined an association between the presence of
the E4 allele with late-onset (greater than 60
years of age) Alzheimer's disease (115, 116).
Furthermore, a gene-dose response exists for
the E4 allele and the risk of late-onset Alzhei-
mer's. In one case-control study, the odds ratio
for developing Alzheimer's disease was 3.9 for
the E3lE4 genotype and 15.6 for the E41E4
genotype. Interestingly, the allelic variant of
APOE also influences treatment response
with the cholinesterase inhibitor, tacrine
(117). In one study, 83% of non-APOE4 carry-
ing patients had improvements in cognition
when given tacrine. In contrast, 60% of pa-
tients with an E4 allele were unchanged or
declined after tacrine administration. Despite
these data, single SNP studies of the APOE
 4 Pharmacogenomics
gene would not justify withholding therapy
because 40% of patients with the E4 allele had
a positive response to tacrine (117). Future
studies will need to incorporate a more
genomic approach by examining other puta-
tive polymorphisms involved in response to
pharmacologic therapy for Alzheimer's dis-
ease.
4.4.3 Hypercholesterolemia. Several stud-
ies have identified genetic polymorphisms in-
fluencing the clinical response to HMG-CoA
reductase inhibitors (subsequently referred to
as statins) (118-122).Rather than focusing on
polymorphisms in drug targets, these studies
examined polymorphisms in genes believed to
influence the progression of atherosclerosis.
All studies were randomized, double-blinded,
and placebo-controlled, and are summarized
in Table 12.8. Taken together, the results of
these studies identify genetic subgroups of
placebo-treated patients who have an in-
creased risk of developing major coronary
events. Furthermore, treatment with a statin
abolished the deleterious effect of the genetic
variant.
Three pharmacogenomic studies have been
published from the Regression Growth Evalu-
ation StatinStudy (REGRESS) group. REGRESS
examined the ability of pravastatin to retard
I
I progression of atherosclerosis among men
with symptomatic coronary artery disease and
hypercholesterolemia (123). In the first exam-
ple, DNA was examined for a polymorphism in
the gene encoding cholesteryl ester transfer
protein (CETP), which is involved in the me-
tabolism of high-density lipoprotein (HDL), a
cardioprotective lipoprotein (122). The pres-
ence of the variation in CETP was referred to
as B1 and its absence as B2. Response to prav-
astatin and placebo was examined by genotype
with patients grouped as BlB1, BlB2, or
B2B2. At baseline, patients with the BlBl ge-
notype had higher CETP and lower HDL con-
centrations than patients with the B2B2 geno-
type. Placebo-treated patients with the BlBl
genotype experienced the greatest progression
of atherosclerosis, the BIB2 genotype had an
intermediate progression, and the B2B2 group
had the least disease progression. Pravasta-
tin-treated patients with either the BlBl or
BIB2 genotype had significantly less athero-
sclerotic progression compared with patients
receiving placebo. However, pravastatin-
treated patients with the B2B2 genotype,
(16% of the study population) derived no ben-
efit from pravastatin treatment as measured
by changes in mean luminal diameter of the
coronary arteries. Although these results are
provocative, a major limitation is that they do
not examine hard clinical endpoints such as
death and non-fatal myocardial infarction.
Plaque composition, rather than size, is a bet-
ter predictor of lesions susceptible to rupture
resulting in an acute coronary event.
A second substudy from REGRESS exam-
ined a polymorphism in the promoter region of
the stromelysin-1 gene [-I612 (5N6A)I (120).
Stromelysin-1 is involved in connective tissue
remodeling and wound healing. The 6A allele
results in reduced expression of stromelysin-1
and consequently increases connective tissue
deposition and potentially increased athero-
sclerotic lesions. Patients were divided accord-
ing to genotype (5A5A, 5A6A, and 6A6A).
There were no differences in prognostic base-
line characteristics, disease severity, or lipid
values among the three genotypes. Further-
more, there was no association between the
stromelysin-1 polymorphism and changes in
lipid levels from pravastatin therapy. Placebo-
treated patients with at least one 6A allele had
the greatest number of clinical events (mostly
restenosis). Pravastatin therapy reduced clin-
ical events most effectively among 6A carriers.
A third pharmacogenomic substudy of
REGRESS examined the impact of the -455
G/A polymorphism of the p-fibrinogen gene
(119). Previous work revealed that carriers of
the -455 A allele have higher concentrations
of fibrinogen, a prothrombotic substance asso-
ciated with increased risk of myocardial in-
farction and stroke (124).At baseline, patients
with the -455 AA genotype had greater ath-
erosclerotic disease severity compared with
those with other genotypes. During follow-up,
placebo-treated patients with the -455 AA ge-
notype had the greatest disease progression.
Once again, the effect of genotype on disease
progression was abolished by pravastatin
therapy.
The Cholesterol and Recurrent Events
(CARE) trial enrolled patients with a history
of myocardial infarction and hypercholesterol-
Table 12.8 Polymorphisms Influencing Clinical Response to Statins
FU Placebo Event Statin Event
Study (Statin) N Orears) Polymorphism Studied Endpoint Rate Rate P
-
- -- ~ p~

Apolipoprotein E (E4, E3, Mortality E4 Carriers; RR E4 Carriers; RR NR

E2) 1.9 (95%CI 0.33 (95% CI
1.13.1) 0.1M.7)
Without E4;
RR 0.66 (95%
CI 0.35-1.24)
REGRESS (pravastatin) 496 2 Stromelysin-1(-1612 5A/6A) Clinical events 5A5A = 17% 0.038
(120) 5A6A = 9%
6A6A = 14%
REGRESS (pravastatin) 807 2 CETP (B1 or B2) Average decrease in mean BIB1 = 0.05 0.01
(122) coronary diameter BIB2 = 0.07
(mm) B2B2 = 0.09
REGRESS (pravastatin) 682 2 p-Fibrinogen (-455 GIA) Average decrease in mean GG = 0.07 0.024
(119) segment diameter (mm) GA = 0.05
AA = -0.06
CARE (pravastatin) 767 5 GP IIIa (PlA',PlA2) CV death, non-fatal MI PlAlA2;RR 0.69 NR
(118) (P= 0.06)

FU, follow-up; P, interaction between placebo and statin therapy; NR, not reported; RR, relative risk; CI, confidence interval; 4S, Scandinavian Simvastatin Survival Study;
REGRESS, Regression Growth Evaluation Statin Study; CARE, Cholesterol and Recurrent Events; CETP, Cholesteryl ester transfer protein; GP, glycoprotein; CV, cardiovascular;
MI, myocardial infarction.
5 Research and Development
emia (125). Overall, pravastatin therapy for 5
years reduced the risk of fatal coronary heart
disease and non-fatal myocardial infarction. A
substudy of CARE focused on whether poly-
morphism~in the glycoprotein (GP) IIIa gene
and the ACE I/D polymorphism were associ-
ated with the reduction in the primary end-
point of the study. The investigators found
that the largest benefit of pravastatin treat-
ment in reducing the primary endpoint oc-
curred in patients with the GPIIIa P1AlIA2 ge-
notype who also carried at least one D allele of
the ACE gene (118).
Finally, a pharmacogenomic study of the
Scandinavian Simvastatin Survival Study
(4s) investigated the impact of the apolipopro-
tein E4 allele with the prognosis and treat-
ment response to either simvastatin or pla-
cebo (121). Among myocardial infarction
survivors who received placebo and had at
least one apolipoprotein E4 allele, the relative
risk for all cause mortality was 1.9 (95% CI,
1.1-3.1). The detrimental impact of the E4
allele was not evident among patients who
received simvastatin (RR 0.33; 95% CI,
0.16-0.69).
Taken together, the results of these five
studies identify genetic subgroups of placebo-
treated patients who have an increased risk of
developing major coronary events. In all stud-
ies, treatment with a statin abolished the
harmful effect associated with the genetic
variant. These results support the observation
that, in general, high-risk patients with isch-
emic heart disease derive the largest relative
benefit from treatment. However, whether
current recommendations regarding choles-
terol-lowering therapy are applicable to pa-
tients in certain genetic subgroups is uncer-
tain. At this time it is premature
- to withhold
statin therapy from any patient meeting crite-
ria for treatment based on national consensus
guidelines. Future prospective genetic epide-
miology studies may further delineate the role
of genetic variants on the development and
progression of coronary artery disease and re-
sponse to treatment.
4.5 Clinical Relevance
Historically, pharmacogenetics targeted its
pursuit on the reasons for toxicity and there-
fore drug safety. However, it is pharmaco-
genomics that promises to deliver individual-
ized pharmacotherapy with greater efficacy
(5) while still limiting toxicity. An example of
the potential for improved efficacy is given in
the example of clozapine. Clozapine is an atyp-
ical antipsychotic with superior efficacy in pa-
tients with treatment resistant schizophrenia.
Despite being an effective anti-psychotic, clo-
zapine therapy is limited by the serious
adverse effects of tachycardia, orthostatic hy-
potension, and agranulocytosis. In fact, agran-
ulocytosis is such a severe and worrisome ad-
verse event that it has necessitated a national
registry to track and monitor patients receiv-
ing clozapine therapy. In addition, large inter-
individual response exists, with only 30-60%
of patients responding to clozapine. A retro-
spective study examined 19 genetic polymor-
phism~in 10 different genes with response to
clozapine in 200 Caucasian schizophrenic pa-
tients (126). The results revealed that a com-
bination of six polymorphisms resulted in a
76.7% prediction of treatment success with
clozapine. The combination of polymorphisms
had a sensitivity of 95% for identifying pa-
tients with a beneficial response to clozapine.
It remains to be determined whether this com-
bination of polymorphisms retains its high
predictive value in a population of diverse eth-
nic backgrounds. However, this study should
serve as a model for future prospective studies
to incorporate a more genomic andlor haplo-
typic approach when attempting to correlate
genetic variability with drug response.
5 RESEARCH A N D DEVELOPMENT
The numbers are staggering. It costs the phar-
maceutical industry approximately $880 mil-
lion and 15 years to go from target identifica-
tion through regulatory approval for a novel
drug. One-half of this cost and time occur dur-
ing phase 11-111 clinical trials. Contributing to
the prodigious cost and time are the many in-
efficiencies of drug discovery, development,
and clinical trials. Seventy-five percent of the
costs of drug development are incurred from
late-stage clinical trials. Incorporation of
pharmacogenomicdata may lead to a dramatic
change in the way clinical trials are designed
and conducted. Pharmacogenomics may re-
642 SNPs: Single Nucleotide Polymorphisms and Phar.macogenomics-Individually
Table 12.9 Recent Drugs
RemovedlRestricted by the FDA
Alosteron
Astemizole
Bromfenac
Cerivastatin
Cisapride
Felbamate
Grapafloxacin
Mibefradil
Rapacuronium
Dexfenfluramine
Terfenadine
Troglitazone
Trovafloxacin
sult in more efficient trials that would be as-
sociated with a lower cost to bring a new chem-
ical entity to market. Moreover, this could
result in shorter time to drug approval, longer
patent protection, and more importantly, in-
creased time of market exclusivity. A recent
report concluded that incorporation of genomic
technologies currently available could result
in savings of up to $300 million per novel drug
and cut 2 years form the drug development
process (www.bcg.com). This section will fo-
cus on specific examples of how pharmaco-
genomic data can be incorporated into clinical
trials to identify responders and exclude non-
responders to reduce unnecessary adverse
events.
5.1 Influence of Pharmacogenomics on
Clinical Trials
In recent years, the complexity and cost of
clinical trials has increased. It is not uncom-
mon for phase I11 drug studies to involve thou-
sands of patients with several years of follow-
up. However, the results of these trials only
provide information on the average treatment
effect in a population, not for an individual
patient. In addition, numerous drugs advance
all the way to phase I11 trials only then failing
to demonstrate any treatment benefit or hav-
ing an unacceptable adverse event profile that
prevents regulatory approval. Recent years
have also seen several promising drugs ap-
proved by the FDA and then withdrawn from
commerce or restricted secondary to serious
adverse events (Table 12.9).
Designed Drug Therapy
5.2 Use of Pharmacogenomic Data in the
Clinical Trial Process
Assume that a company has a novel drug to
treat chronic heart failure in phase I1 trials.
Data from this study demonstrates that 35%
of the study population receiving active drug
achieves a therapeutic response compared
with 15% of placebo-treated patients. Do these
data warrant further study? If this hypotheti-
cal phase I1 trial had incorporated pharmaco-
genomic data, the potential exists that a ge-
netic subgroup of responders could have been
identified. Suppose that 60% of patients with
the hypothetical AA genotype respond to this
drug while patients with genotypes Aa and aa
have a response rate similar to placebo. A ho-
mogeneous patient population (AA genotype)
likely to respond to the medication could now
be selected for a confirmatory phase I11 trial.
The sample size needed for a phase I11 trial
would be reduced because patients likely to
respond to the medication have been enrolled.
In fact, the majority of clinical trials that use
pharmacogenomic information will require
smaller sample sizes compared with trials in
which no genotypic information is collected
(127).
Alternatively, assume a company has de-
veloped an investigational drug to treat type 2
diabetes mellitus. Phase I1 studies demoh-
strate that 75% patients reduce their glycosy-
lated hemoglobin (HbA,,) levels by 1.5%.
However, the drug is associated with a serious
adverse event in 5% of patients. What impact
will this serious adverse drug event have on
the approval and use of this agent? If DNA
samples had been collected from all study par-
ticipants, a population of patients predisposed
to developing this toxicity may have been iden-
tified. The susceptible patient population
could then be excluded from phase I11 trials,
and assuming that the agent is eventually ap-
proved, a bedside molecular diagnostic test
could be used to prospectively identify pa-
tients predisposed to the serious adverse
event. In this aforementioned example, every-
one benefits, including the drug manufac-
turer, patients, and the health care system by
avoiding the costs of a serious adverse drug
event. Furthermore, pharmacogenomic data
could also be used in post-marketing surveil-
5 Research and Development
lance, which would dramatically improve our
current surveillance system (128).
Four pieces of information are important
before planning a clinical trial using pharma-
cogenomic data (127). First, the allele fre-
quency for the polymorphism of interest must
be known. Most trials with a nominal outcome
variable would require sample sizes of greater
than 1000 patients for allele frequencies of
less than 10%. For these rare SNPs, associa-
tion with efficacy and or toxicity might only be
discerned after the drug has been approved.
However, studies of polymorphisms with an
allele frequency of 30-50% would require
fewer subjects compared with a trial that does
not use pharmacogenomic information (127).
Second, the gene action of the SNP must be
understood. Does the SNP behave in a domi-
nant, additive, or recessive fashion? A domi-
nant action means that the genotype Aa dis-
plays the same phenotype as genotype AA.
This example would require fewer study par-
ticipants compared with alleles displaying ad-
ditive or recessive action. Third, the investiga-
tor should have knowledge of genotype
relative risk (GRR). This is potentially the
most difficult of the factors to have a ~ r i o r i
A
knowledge, and may require assumptions, as
are typically done to estimate the sample size
of most clinical trials. As with any trial, the
smaller the genotype relative risk, the larger
the number of subjects required. The GRR is
likely to be small given the multitude of fac-
tors influencing drug response. Fourth, as the
number of alleles tested increases, the sample
size also must increase. For example, studying
10 loci would require a sample size 1.5 times
larger than studying a single locus. Studies of
100 loci would require sample sizes twice as
large compared with a single locus. Studying
100,000 loci necessitates a sample size three
times larger compared with a single locus (127).
5.3 Examples from the Literature
A recent study prospectively genotyped all pa-
tients for the CYP2D6 gene and excluded poor
metabolizers to enhance patient safety (129).
The study was a randomized, double-blinded
comparison of lamotrigine with desipramine
in patients with unipolar depression. Desipra-
mine is a substrate for CYP2D6 and poor me-
tabolizers of this enzyme have serum desipra-
mine concentrations markedly higher than
extensive metabolizers (31). In all, 6.1% of
subjects screened were excluded from the trial
because they were identified as poor metabo-
lizers. Clearly, using genotyping as an inclu-
sion or exclusion criterion requires that the
genotype(s1of interest can be determined in a
rapid fashion. For trials that involve acute
treatment, a point of care or "bedside" test
would need to be developed to potentially ex-
clude patients at an increased risk of adverse
events.
Currently, 18 of the top 20 pharmaceutical
companies are collecting DNA data in phase I1
and I11 trials. Among them is GlaxoSmith-
Kline, which has added this component to
studies in all major therapeutic areas of their
drug development process (130). A survey
from the SNP consortium estimates that
within 5 years, 50% of clinical trials will in-
volve genotyping. Unfortunately, in many
cases, DNA collection is an optional part of the
protocol. Reasons for this include lack of
knowledge from the investigators or the per-
ception that collection of genomic DNA from
patients will lead to delays in Investigational
Review Board (IRB) approval or patient re-
cruitment.
It is understandable why the pharmaceuti-
cal industry may have some trepidation of .
using pharmacogenomics in the clinical trial
process. The basic tenet of most of the phar-
maceutical industry has been to develop drugs
in a one-size-fits-all approach with the hope
that they will become blockbusters, generat-
ing greater than $1 billion in annual sales.
There has been some concern that pharmaco-
genomic data will reduce the market share of a
drug by identifymg the subgroups of patients
who actually derive therapeutic benefit from a
drug. Paradoxically though, integration of
pharmacogenomics could actually increase
market share for a drug. The goal for any new
product is to capture 100% of the target mar-
ket; however, for a drug, the target market is
not composed of all patients with the particu-
lar disease that the drug is indicated to treat.
Consider the following example. A pharma-
ceutical company develops a new drug to treat
hypertension. What will be the market pene-
tration for this new antihypertensive agent in
the first year after regulatory approval? Hy-
644 SNPs: Single Nucleotide Polyrnorphisrns and Pharrnacogenornics-Individually Designed Drug Therapy
pertension affects approximately 50 million
Americans, but several classes of medications
such as P-blockers, angiotensin-converting
enzyme inhibitors, and angiotensin receptor
blockers exist, with numerous drugs in each
class. Consequently, it is difficult for any new
drug in each of these classes to acquire a large
market share. However, what if anew agent in
one of these classes was approved with a mo-
lecular diagnostic to predict efficacy. A third
or fourth in class drug approved with a diag-
nostic assay could provide a competitive niche
demonstrating a high response rate in selected
groups and a low adverse event rate in other
groups. It is likely in this scenario that the
market penetration as well as market share
achievement will be greater if this drug is cou-
pled with a molecular diagnostic test. Thus
pharmacogenomic data could be an im-
mensely useful marketing strategy for a drug
company.
Several major unanswered questions exist
as to how the FDA will react to new drug ap-
plications that contain pharmacogenomic
data. Clearly, drugs approved for genetic sub-
groups will require molecular diagnostic test-
ing and thus raise several compelling ques-
tions (131). First, how will this affect off-label
prescribing? What impact will this have on li-
ability? Second, what happens if a patient re-
fuses to have a molecular diagnostic test per-
formed? Are they now limiting themselves to
new therapeutic agents? What happens to the
low socioeconomic populations who have no
insurance and can not afford the cost of the
genetic test? Is this population now excluded
from receiving potentially safe and effective
pharmacotherapy? How will pharmacogenom-
ics influence Orphan Drug status? In the
United States, drugs developed for conditions
affecting less than 200,000 individuals get 7
years of market exclusivity, unless an alterna-
tive medication is proven superior. Will this be
the financial incentive that some companies
require? Compelling questions indeed.
6 OBSTACLES FACING THE FIELD OF
PHARMACOGENOMICS
6.1 Complexity and Cost
Current methods to sequence DNA are too
costly and laborious to allow implementation
into routine clinical practice. The cost to geno-
type one SNP can range anywhere from $1 to
$50, and this prohibits phase 11-111trials from
examining several hundred putative SNPs per
patient that influence drug toxicity or thera-
peutic benefit. An alternative approach would
be to examine approximately 10-15 SNPs in
candidate genes that are involved in the drug
targets, drug transport, and metabolism.
However, the candidate gene approach may
not be plausible during initial drug discovery
and development. Advances in high-through-
put technology and increased competition
should eventually reduce the cost of genotyp-
ing SNPs to $O.OOl/SNP, thus making it viable
to evaluate 100,000-200,000 SNPs per pa-
tient. Another factor contributing to cost in-
volves the initial investment in the equipment
and technology required to fully integrate
pharmacogenomicsinto all aspects of drug dis-
covery and development. However, these up-
front costs may result in significant cost sav-
ings by increasing efficiency of clinical trials
with the identification and termination of
drugs with little potential of eventual regu-
latory approval. The deluge of the data gen-
erated from pharmacogenomic studies also
requires advances in bioinformatics and
data mining strategies. The extent to which
pharmacogenomics pervades clinical practice
is largely
- . dependent
- on the ability of bioinfir-
matics to transform the prodigious amounts of
data into knowledge. Consequently, the bioin-
formatics budgets of some pharmaceutical
companies have increased 20-60%. A sure in-
dication of the desperate need for this technol-
ogy.
6.2 Will Pharmacogenomics Improve
Medical Care?
It is still unclear if prospectively genotyping
patients for many of the genetic variants de-
scribed within this chapter improves medial
care and whether it is cost effective. Geno-
typing patients for the presence of TPMT
deficiency to prevent life-threatening hemato-
logical toxicity from azathioprine, mercapto-
purine, or thioguanine provides an equivocal
advantage compared with empirical dosing.
However, in other examples, genotyping may
not be an advantage over the current best
medical care. For example, several studies
6 Obstacles Facing the Field of Pharmacogenornics
have demonstrated that individuals carrying
mutant alleles for CYP2C9 have decreased
metabolism or elimination of warfarin (25,
132-134). One retrospective trial also showed
that these individuals have a higher risk for
both minor and major bleeding (25). However,
it remains to be elucidated whether prospec-
tively genotyping patients receiving warfarin
reduces bleeding complications when com-
pared with the best available standard of care.
This question can only be answered through a
randomized controlled trial comparing a
genomic approach with a traditional dosing
approach.
Recently, the Statin Response Examined
by Genetic HAP Markers Study (STRENGTH)
completed enrollment of 600 patients with hy-
perlipidemia. Patients were randomized to
one of four statin treatment regimens (ceri-
vastatin, pravastatin, atorvastatin, or simva-
statin) for a 16-week duration. The goal is to
identify genetic markers that predict which of
the four statins is most beneficial in reducing
cholesterol levels. However, whether this ap-
proach is superior to management of patients
in a specialized lipid clinic remains to be deter-
mined.
6.3 Paradigm Shift in Health Care
Transitioning from the one-size-fits-all ap-
proach to personalized medicine will create a
paradigm shift for both health care providers
and the pharmaceutical industry. There is
some precedence for drugs specifically tar-
geted for a subset of disease. Trastuzumab
(Herceptin) is approved only for the 2530%
women whose breast cancer overexpresses the
HER-2/neu protein. Trastuzumab is marketed
along with a molecular diagnostic, the DAKO
Hercep Test. This test is a semi-quantitative
assay for testing breast tumor tissue that
overexpresses the HER-21neu protein. In its
first year on the market, trastuzumab gener-
ated $188.4 million in sales. Thus, an agent
that probably would not have received FDA
approval is now an effective alternative for a
specific subgroup of women with breast can-
cer.
Physicians will also need to adjust to the
shift from the art of medicine to the science of
medicine. Rather than selecting a drug based
on experience, the selection may be based on
the analysis from a computer program. Rather
than making a diagnosis based on phenotypic
symptoms, a disease may be diagnosed years
before it manifests, based on an individual's
genetic makeup.
6.4 Ethical Considerations
Pharmacogenomics has created a new lexicon
that all health care providers must familiarize
themselves, and thus precise language is fun-
damental when dealing with pharmacogenom-
ics. It is imperative that pharmacogenomics
be distinguished from genetic predisposition
testing. All investigators in the field must con-
vey this concept to the public, members of the
health care team, and the insurance sector. In
the majority of cases, identification of SNPs
to predict drug response carries no prognostic
information for diseases. Determination of
SNPs to predict drug response is analogous to
obtaining culture and sensitivity data to guide
antimicrobial therapy. However, in other
cases the potential for discrimination exists.
One example involves the apolipoprotein E4
allele. Genetic variation in the apolipoprotein
E4 allele may be examined to explain the vari-
ability among statin therapy (121). However,
these results could have significant implica-
tions in predicting the risk of Alzheimer's dis-
ease later in life (115, 116). Clearly, a n enor-
mous potential for discrimination exists if an
insurance carrier discovers the results of this
diagnostic test. As an example, the New Jersey
Genetic Privacy Act, passed in 1996, prevents
employers and insurance carriers from dis-
criminating based on genetic tests. Indeed,
further state and federal legislation like New
Jersey's would help to allay some of these eth-
ical concerns and public fear of genetic testing.
Undeniably, genomics and phannacogenom-
ics opens up a whole host of legal, ethical, and
societal issues that will have implications in
patient confidentiality, discrimination, mal-
practice, and informed consent (135-137).
However, as clinicians, scientists, and health
care practitioners, we must always remember
the unquestionable power of an individual's
right to choose, and prospectively fight to en-
sure patients' rights and prevent genomic dis-
crimination.
646 SNPs: Single Nucleotide Polymorphisms and Pharmacogenomics-Individually Designed Drug Therapy
7 CONCLUSION
The publication of the draft of the Human Ge-
nome Project represents an acmatic scientific
breakthrough. One of the first discernible ben-
efits from the Human Genome Project will be
advances in pharmacogenomics. Pharmaco-
genomics is likely to have a major role in the
daily practice of medicine in the near future.
In many ways, a proof of principle is required.
Although trastuzumab is an effective agent,
its use is based on differences in protein ex-
pression and not genetic variation. It is our
belief that there will be a surge in the field
after the first drug developed through func-
tional genomics becomes FDA approved, or
when the first assay to predict efficacy or tox-
icity of an existing drug is approved. Whatever
our future holds, we must always remember
the ultimate goal of pharmacogenomics: to de-
velop or use truly individualized pharmaco-
therapy that will produce the most benefit and
the least harm thus extending and enhancing
human life.
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CHAPTER THIRTEEN

Plasmid DNA-Mediated Gene

Therapy
RAJKUMARBANERJEE
LEAF HUANG
Center for Pharmacogenetics
School of Pharmacy
University of Pittsburgh
Pittsburgh, Pennsylvania

Burger's Medicinal Chemistry a n d Drug Discovery
Sixth Edition, Volume 4: Autocoids, Diagnostics,
and Drugs from New Biology
Edited by Donald J. Abraham
ISBN 0-471-37030-4 O 2003 John Wiley & Sons, Inc.
651
Contents
1 Introduction, 652
2 Direct Delivery with Naked Plasmid DNA, 652
2.1 History and Recent Therapeutic Uses, 652
2.2 Gene Delivery for Myocardial Diseases, 655
2.3 Gene Therapy for Angiogenesis, 656
2.4 Gene Therapy for Autoimmune Diseases, 656
3 Improving Plasmid DNA-Mediated Gene
Transfer by Electroporation, 656
4 Improving Plasmid DNA Transfer Mediated by
Gene Gun, 657
5 Lipid-Based Vectors, 658
5.1 First Generation of Cationic Lipids, 658
5.2 Cellular Barriers for Transfection, 659
5.2.1 Structure of LipidDNA Complex, 659
5.2.2 Entry of DNA Into Cells, 660
5.2.3 Fate of Complex Inside the Cell:
Escape into Cytosol, 661
5.2.4 Entry of DNA in Nucleus, 662
6 I n Vivo Use of Cationic LiposomeDNA Complex
(Lipoplex), 663
6.1 Intravenous Injection, 663
6.2 Direct Injection, 663
6.3 Intraperitoneal Injection, 664
7 Second Generation of Lipidic Delivery System
(Lipopolyplex), 665
7.1 Polycation-Condensed DNA Entrapped in
Cationic Liposome: LPD-I Formulation, 665
7.2 Polycation Condensed DNA in Anionic
Liposomes: LPD-I1 Formulation, 666
8 Emulsion-Mediated Gene Transfer, 666
8.1 General Development, 666
8.2 Reconstituted Chylomicron Remnants
for Gene Transfer, 667
9 Gene Delivery by Polymeric Systems (Polyplex),
667
652
9.1 General Development and Uses, 667
9.2 Targeted Gene Delivery by Antibodies
Conjugated with Polycations, 669
1 INTRODUCTION
In the post-genome era, many defective genes
responsible for various disease phenotypes
have already been or will soon be discovered.
Introduction of the normal genes effectively
into the target cells and/or tissues to comple-
ment the defective genes is a daunting chal-
lenge to scientists of various disciplines. Gene
therapy methodology was originally designed
to correct inheritable disorders but is now be-
ing considered for treating acquired disorders,
such as cancer and AIDS.
Gene therapy is broadly divided into two
different categories, one that uses viruses and
the one that doesn't. Replication-defective vi-
ruses with apart or the entirety of their coding
sequences replaced by a therapeutic gene are
used to transduce cells with very high effi-
ciency. Retrovirus, adenovirus, adeno-associ-
ated virus, herpes simplex virus, papilloma vi-
rus, sendai virus, etc., have been used for
various gene therapy approaches either clini-
cally or experimentally. Although viral vectors
have shown efficacy in transducing cells and
tissues, they have several drawbacks that
limit their widespread use. For example, ad-
enoviral vector induces strong host immune
responses, which induce toxicity and limit the
duration of the transgene expression. Immu-
nogenicity makes repeated dose impossible.
Retroviral vector requires dividing cells for
gene expression, and it may integrate ran-
domly into the host genome, posing risk for
mutation and neo~lastictransformation. Vi-
ral vectors are also'difficult to produce in large
scale. These concerns have prompted the de-
- -
velopment of non-viral vectors as a less toxic
and more scalable alternative for gene ther-
apy. However, the efficiency of non-viral vec-
tors still needs improvement; so does the du-
ration of the transgene expression. These
aspects are the central concerns of the current
non-viral vector development.
The carrier for the therapeutic gene in non-
viral vectors is often a recombinant plasmid
Plasmid DNA-Mediated Gene Therapy
10 Non-Viral Vector-Related Cytotoxicity, 669
11 Conclusion, 670
12 Acknowledgments, 670
DNA (pDNA). Recombinant DNA is a syn-
thetic DNA made by connecting different
DNA fragments from different sources into
one recombinant molecule, whereas plasmid
DNA is a non-chromosomal, circular, and su-
percoiled bacterial DNA, which is produced in
bacterial cultures by fermentation. A thera-
peutic or a marker gene can be introduced into
pDNA by simple recombinant procedure.
pDNA can be purified by simple physical and
chemical methods. Advanced technologies ex-
ist such that multigram quantities of pDNA
can be routinely produced from a single lot.
Larger scale production can also be done on
demand. Standard pDNA is 4-10 kilobase
pairs in size (MW 1 X lo6 to 3 x lo6). Other
than the bacterial sequences, pDNA for gene
therapy contains a transgene, which is usually
the expressible sequence of a genomic DNA,
popularly known as complementary DNA
(cDNA) that encodes a protein. Transcription
of the transgene to make messenger RNA is
controlled by another gene sequence called
promoter, which locates upstream of the cod-
ing sequence of transgene. To ensure proper
termination of transcription, a transcription
termination gene sequence is often inserted
downstream of the transgene. A polyadenyla-
tion sequence and sometimes an intron, i.e., a
non-expressible gene sequence, is also added
to ensure proper processing of the mRNA
product of the transgene (Fig. 13.1). In this
chapter we will emphasize the usefulness of a
transgene in the form of plasmid DNA associ-
ated with non-viral vectors to decipher various
biological functions and for development of
gene therapy.
2 DIRECT DELIVERY WITH NAKED
PLASMID DNA
2.1 History and Recent Therapeutic Uses
The simplest of all DNA delivery systems is
the injection of naked pDNA to the organ or
tissue of interest. Intramuscular injection of
2 Direct Delivery with Naked Plasmid DNA
Figure 13.1. Schematic representation of a typical
plasmid DNA. Promoter, promoter sequence; in-
tron, intron sequence; cDNA, complementary DNA
encoding a gene; poly A, poly-adenylation sequence;
termn, termination sequence.
naked pDNA was the first instance where the
skeletal muscle was transfected (1). Since
then, a variety of tissues had been transfected
with direct gene transfer. Naked pDNA had
been injected into the interstitial space of liver
(2-41, thyroid (51, heart muscles ( 6 ) ,brain (71,
and urological organs (8). The naked DNA
containing one or more anticancer genes were
also injected in various tumors with mixed ef-
ficacy (9-11).
This technique of direct injection of pDNA
into an intact organ had become a useful tool
to analyze the gene expression and promoter
function in the respective organ. By the use of
a viral promoter, such as that from cytomega-
lovirus (CMV), a high level of expression was
attained initially with pDNA vectors. How-
ever, the expression declined sharply to near
background within 2-3 weeks. Herweijer et al.
(3) have shown that, although the maximum
decrease in the expression level occurred
within 2 days from pDNA transfer, pDNA
could be detected for at least 12 weeks after
injection (0.2 copies per genome). Promoter
activation associated with the immune re-
sponses raised against the expressed trans-
gene product was believed to be the major
cause of this early decline in gene-expression
level. This fact is corroborated in immunosup-
pressed mice that showed extended level of
transgene expressions.
To sustain therapeutic response initiated
by the transfection of pDNA containing ther-
apeutic gene, it is necessary to ensure a long-
lasting gene expression eliciting little to no ex-
pression-related cytotoxicity. Towards this
end, few gene transfer techniques can sustain
transgene expression over an period of time.
Mouse liver transferred with high-expressing
human factor M (hFIX) pDNA yielded thera-
peutic-level gene expression over 1.5 years,
eliciting no expression-related or therapy-re-
lated toxicities (4). A declined transgene ex-
pression observed after liver regeneration fur-
ther suggested that the maintenance of the
plasmid had declined, rather than the trans-
gene being integrated into the host genome.
Hence, sustained gene expressions required a
transcriptionally active vector DNA that could
persist for long time in the organ. These re-
sults demonstrated that non-viral plasmid
transfer could lead to extended life of trans-
gene-expression level without genome inte-
gration.
Non-viral gene therapy had become a use-
ful tool in generating various therapeutic ef-
fects in diverse animal models. One of these .
effects is to induce anticancer response in tu-
mor models by introducing anticancer genes.
In an effort to elicit anticancer effect in tu-
morogenic cells implanted in medullary thy-
roid cancer (MTC) rat-xenografts, NO syn-
thase I1 (NOS 11)gene, which catalyzes nitric
oxide (NO) production, was incorporated in a
plasmid under the control of C M V promoter
and injected in the rat. The gene triggered a
suicidal effect (apoptc .is) on the tumor cells
by the production of NO, which specifically ac-
tivated the macrophages against the fast di-
viding tumor cells, leaving normal cells un-
touched (9). Most importantly, the activated
NOS gene in a tumor cell induced the cytotox-
icity in the neighboring tumor cells, i.e., an
NO-mediated bystander antitumor effect was
also observed (10). The fact that nitric oxide
mediated tumorogenic effect doesn't require
transfection of all neoplastic cells promised a
capable suicide gene therapy approach for hu-
man cancer.
654
Similar bystander effect could be obtained
by other apoptosis-inducing molecules such as
Fas ligand (FasL). FasL and its receptor Fas
are membrane proteins, which on mutual in-
teraction initiate an apoptotic signal in Fas-
bearing cells. pDNA could be used to deliver
these apoptosis-inducing gene to initiate kill-
ing of transfected and non-transfected sur-
rounding cells. On direct injection of FasL-en-
coding pDNA vector into the inflamed thyroid
(12), pathogenic lymphocytes were inhibited
to enter into thyroid leaving the already-infil-
trated T-cells dead. Thus, FasL expressing in
thyroicytes might lead to potential remedial
therapy for the experimental autoimmune
thyroiditis (EAT).
Direct pDNA transfer technique had been
used to examine the role of a physiologically
related protein transduction signal pathway
toward certain endogenous disease pheno-
type, such as investigating the function of the
tissue kallikrein-kinin system (KKS) in the
central control of blood pressure homeostasis
(13). Kallikrein is a proteinase enzyme, which
converts kininogen to vasodilative kinin pep-
tides. The human tissue kallikrein gene, in the
form of naked pDNA (CMV-cHK),was directly
delivered by intracerebroventricular injection
into hypertensive rats. The expression of hu-
man tissue kallikrein protein was identified in
the cortex, cerebellum, brain stem, hippocam-
pus, and hypothalamus of the treated rats.
The expression level and its effect could lead to
understanding the role of vasodilative KKS on
the pathogenesis of hypertension.
Direct gene transfer to skeletal muscle was
mainly used for the treatment of variety of
diseases, e.g., muscular dystrophies, chronic
ischemic limb syndromes, etc. Specifically en-
gineered pDNA and the vector systems were
developed that enabled regulated and tissue-
specific transgene expression in skeletal mus-
cle in vivo (14).Naked pDNA based gene
transfer (1,15) has been used in different an-
imal models, e.g., in correcting Duchenne
Muscular Dystrophy (16), to supply sources of
therapeutic protein systemically (17, 181, for
genetic-vaccination against pathogens (19)
and tumor cells (20,21). However, clinical use
of this technique is limited because of ineffi-
cient gene expression specifically in large ani-
mals.
Plasmid DNA-Mediated Gene Therapy
On quantifying the gene uptake in the mus-
cles, the intramuscular injection showed less
than 1%uptake of injected dose and was lim-
ited to cells adjacent to the needle track (22).
Using hypertonic sucrose (23) or muscle revi-
talizers such as bupivacaine (24, 25), the effi-
ciency and reproducibility of gene expression
could be increased. A 100-fold higher level of
transgene expression throughout the muscles
of hindlimb was observed on intra-arterial in-
jection of naked pDNA into the femoral arter-
ies of rats than direct intramuscular injection
(16). Myofibers were 10% more transfected
through intravascular delivery than with the
direct intramuscular injection. Zhang et al.
(26) extended the same intra-arterial injection
technique to the non-human primates. It
could be hypothesized that pDNA was extrav-
asated by the intravascular pressure during
the injection, most likely by convective flow
across the endothelium (27, 28). Once the
pDNA is extravascular, the muscle cells in situ
with the help of a receptor mediated uptake
process pick up the naked pDNA. This was
evidenced by the fact that the naked pDNA
was taken up by hepatocytes in vivo by a re-
ceptor-mediated process (28). Several DNA re-
ceptors had been discovered in human leuko-
cytes (291, peritoneal macrophages (301, and
in wide variety of tissues and tumor cells (31-
35). At this point, the poorly understood mech-
anism of molecular recognition and character-
ization of the cell surface receptor(s) involved
in the binding and internalization of DNA
need a fresh look. Liu et al. (36) and Zhang et
al. (37) reported that rapid injection of pDNA
in a large volume (e.g., 5 pg of DNA120 g
mouse in 1.5-2.0 mL of saline solution)
through the tail vein left the injected DNA in
the inferior vena cava. The DNA flowed back
to the tissues linked to this vascular system,
primarily the liver. The hydrodynamic pres-
sure forced DNA into the liver cells before it
was mixed with blood. By this process, the
liver showed the highest expression of gene;
internal organs like lung, spleen, heart, and
kidney were also efficiently transfected. Our
lab had recently shown that briefly clamping
the vena cava following tail vein injection of
pDNA in a small volume efficiently trans-
fected both liver (38) and diaphragm (39). The
result is potentially important because dia-
2 Direct Delivery with Naked Plasmid DNA
phragm is barely transfected by hydrodynam-
ics-based method. The full-length dystrophin
cDNA can be delivered to the diaphragm for
the treatment of Duchenne Muscular Dystro-
phy. It is well known that patients with Du-
chenne Muscular Dystrophy often suffer from
fatal respiratory failure caused by the dystro-
phic diaphragm muscle.
2.2 Gene Delivery for Myocardial Diseases
Naked pDNA transfer into myocardium
through direct injection or through coronary
vasculature usually showed low transfection
efficiencies. The results had nevertheless
proven valuable in studies aimed at character-
izing the role of promoters in cardiac tissue
and for examining the influences of naturally
occurring mechanical and hormonal stimuli of
the myocardium on expression of transferred
foreign genes (40).
Kitsis et al. (41) have demonstrated that
the tissue-specific promoter chimeras injected
into the heart could respond accurately to
shift in thyroid hormone levels in vivo. Injec-
tion of pDNA with gene constructs driven by
cellular promoters resulted in detectable lev-
els of reporter gene activities. The cellular pro-
moter was derived from the rat a-myosin
heavy.chain (a-MHC) gene whose expression
in vivo is restricted to cardiac muscle and is
positively regulated by thyroid hormone. This
method proved valuable to identify the regu-
latory portion of genes expressing specifically
in cardiac muscles. Direct DNA injection had
been extended to evaluate and characterize
the activation properties of a cardiac-specific
promoter/enhancer of the slow/cardiac tropo-
nin C (cTnC) gene that express in cardiac stri-
ated muscles (42). Myocardial direct DNA
injection was also used to analyze the tran-
scriptional regulation of brain creatine kinase
(BCK) gene in the heart (43). pDNA con-
structs containing BCK promoter and CAT or
luciferase reporter gene was delivered into the
left lateral wall and apex of the ventricle on
the heart. The study might provide insight
into the embryonic gene expressing mecha-
nism during cardiogenesis. Because the BCK
gene, the major gene for cytoplasmic creatine
kinase expressed in the embryonic heart, is
down-regulated during cardiogenesis, it is re-
instated in response to stimuli such as isch-
emia, hypertrophy, or heart failure in the
adult.
The method of direct pDNA injection was
used to explore the effect of specific patho-
physiological state on cardiac gene expression,
such as ischemia (44), myocardial infarction,
reperfusion injury, hypertension (45,46), etc.
Ischemia is a disease state formed when tis-
sues are starved for blood supply and nutri-
ents because of deficient supply of blood
through possibly narrowed or blocked arter-
ies. Sporadic myocardial ischemia is com-
monly associated with coronary arterial dis-
eases. To eliminate the ischemia related
disease phenotype, a therapeutic gene is re-
quired, which could be selectively up-regu-
lated by the signals related to the heightened
period of ischemic activity and consequently
down regulated when the activity subdues. In
this context, Prentice et al. (44) introduced
expression plasmids containing muscle-spe-
cific a-MHC promoters and hypoxia-respon-
sive enhancer (HRE) elements linked to a re-
porter gene in cultured cells or into the rabbit
myocardium and measured the regulation of
these constructs by hypoxia or experimental
ischemia. It was shown that the expression of
reporter gene was induced by both hypoxia in
vitro and by a short interval of ischemia in
vivo.
There were different reports concerning
the stability of plasmid-based transgenes in
both skeletal and cardiac muscles. Lin et al.
(47) have shown that the rat cardiac myocytes
could express /3-galactosidase gene under the
control of the Rous sarcoma virus promoter by
the injection of pDNA encoding the reporter
gene directly into the left ventricular wall.
0-galactosidaseexpressed in cardiac myocytes
was detected in rat hearts for at least 4 weeks
after injection of the p-galactosidase gene. In
post-mitotic cardiac and skeletal muscle cells,
the transgene expression of the pDNA de-
clines with time probably because of the episo-
mal localization of the DNA (16,47). The rea-
son that the striated muscles showed higher
capacity of uptake and expressing pDNA fol-
lowing direct injection was not clear; the effi-
cient gene transfer might be induced by cellu-
 Plasmid DNA-Mediated Gene Therapy

lar membrane rupturing and destabilization and organs. Inflammation is a prevalent symp-
followedby inflammation caused by the injec- tom for this disease caused by the excessive
7

tion needle (48). presence of large number of immune cells and

molecules in the target site of the body.
2.3 Gene Therapy for Angiogenesis Against autoimmune diseases or other inflam-
matory conditions, the delivery of cytokines or
Because ischemia is formed because of a defi-
cytokine inhibitors through gene therapy is
cient supply of blood to tissues, formation of
proved very effective. Interferon y (IFN-y), in-
new blood vessels is curative against this dis-
terleukin-1 (IL-1, a or p), IL-12, and tumor
ease. One of the mechanisms that involve the
necrosis factor a (TNFa) are the most fre-
formation of new blood vessels is angiogenesis.
quently addressed inflammatory cytokines in
Angiogenesis is the process of new blood vessel
illness related to autoimmune/inflammatory
development for the vascularization of various
diseases. Other than these cytokines, trans-
organs, for wound healing (49) and to allow
forming growth factor p (TGF P ) is also a key
cancer development and proliferation (50).
regulatory cytokine (53), because TGF p in-
Therapeutic angiogenesis involves restock-
hibits T- and B-cell responses, dysregulation
ing angiogenic growth factors by administer-
of which lead to elevation of autoimmune dis-
ing recombinant proteins or endothelial
ease conditions.
growth factor gene. The recombinant proteins
In animal models, pDNA constructs with
have severe limitation on its usage because it
the encoding anti-inflammatory cytokine
is expensive and difficult for large-scale pro-
genes for IL-10 (54), IL-4 (55), and TGF p l
duction. On the other hand, gene therapy pro-
(56) were injected into either tibialis anterior
vides a systemic and long-term effect with
or rectus femoris muscles in nonobese diabetic
modification in the effective dosage of the
(NOD) mouse against autoimmune diabetic
therapeutic agent. To evade potential problem
of pathological angiogenesis, transient gene
disease. Although- there was no marked de-
crease in severity of insulitis, the diabetes was
expression is usually preferred for this kind of
reduced in NOD mice injected with ILlO com-
treatment. Tsurumi et al. (51) introduced na-
pared with nontreated NOD mice. In another
ked pDNA encoding vascular endothelial
experiment, treatment of autoimmunity
growth factor (VEGF) by intramuscular (IM)
prone NOD mice with pCMV-TGF-pl(57)re-
injection into ischemic hind limb muscles of a
sulted in considerable elevation of TGF-p 1
rabbit model and observed that the vessels
level in the plasma. The increased levels of
and blood-capillaries were increased in rabbit
TGF-p1 exerted various immunosuppressive
muscles injected with VEGF compared with
effects such as there was suppression of de-
controls. An enhanced vascularity-induced
layed-type hypersensitivity (DTH) and pre-
perfusion followed by increased blood flow in
vention of insulitic and diabetic incidence in
the ischemic limbs was also observed. In clin-
this kind of mice. TGF-P1, IL-4, and IFN-y
ical trial, Simovic et al. (52) introduced naked
gene coding plasmid vectors were also injected
pDNA encoding human VEGF gene by direct
IM to rodent models for treatingexperimental
intramuscular injection to chronic ischemic
allergic encephalomyelitis (EAE) (57), sys-
limbs of patients to treat peripheral neuropa-
temic lupus erythematosus (SLE) (58), colitis
thy caused by critical limb ischemia. The pa-
(591, and streptococcal cell wall-induced ar-
tients showed decreased neuropathic disabil-
thritis (SCW-arthritis) (60).
ity in the treated limbs, indicating that a long-
term therapy might improve the integrity in
tissues of ischemic limb and consequent re-
3 IMPROVING PLASMID DNA-MEDIATED
trieval of limb.
GENE TRANSFER BY ELECTROPORATION
2.4 Gene Therapy for Autoimmune Diseases
Electroporation is a process of exposing cells
Autoimmune disease is a pathogenic condition to a controlled electric field for the purpose of
in which one's immune system mistakenly cellular membrane permeabilization (61). The
targets and attacks person's own cells, tissues, intense localized electric pulses destabilize the

4 Improving Plasmid DNA Transfer Mediated by Gene Gun
membrane allowing molecules, which other-
wise do not gain access inside the cell, to enter
cells. A variety of genetic materials were in-
serted into the cells in vitro by electroporation
(62-64). Electrochemotherapy, a cell elec-
tropermeabilization approach that facilitates
the cellular entry of hydrophilic anticancer
agents such as bleomycin, was used to obtain
drastically improved antitumor effect (65) in
malignant melanoma.
Recently, in vivo electroporation has
emerged as a leading technology for develop-
ing non-viral gene therapies and nucleic acid
vaccines (NAV). Naked pDNA injections ac-
companied by electroporation showed 10- to
1000-fold increases in gene expression com-
pared with the same injections without elec-
troporation (66-68), and the duration of a
gene expression after electroporation in vivo
was dependent on target tissue and plasmid
constructs (66). A broader variety of cells
showed reporter gene expression (66-69).
One of the advantages of electroporation is
that specially designed electrode directed
against a given tissue could provide a specific
gene expression for the treated tissue. A high
level of controlled gene expression was ob-
tained by using a tissue-specific promoter di-
rected against a given tissue with different
pulsing parameters (66).
The electroporation-mediated in uivo gene
transfer had been successfully used for hepatic
parenchyma (70, 71), hepatocellular carci-
noma (72), skin (73, 74), skeletal muscle (69,
75,761, mouse testes (771, melanoma (65), hu-
man primary myoblast (78), glomeruli (79),
brain (go), human primary hematopoietic
stem cells (811, human esophageal tumor (82),
and rat skeletal muscle for correcting anemia
of renal failure (83).
Electroporation requires a high electric
field strength, which eventually restricts its
use because it induces some tissue damage.
Efficient and safe electro-transfer for deliver-
ing exogenous material into tissues must be
developed before the clinical potential of gene
therapy can be realized. Our laboratory had
recently developed a novel design of a syringe
electrode, with which electric field was di-
rectly delivered to the cells where pDNA had
been iniected. and the iniected DNA could be
confined to the high field region. High trans-
- - - -
657
fection efficiency was observed with compara-
tively lower field strength, which incurred
minimal tissue damage (Liu and Huang, un-
published observations).
A technique called microelectroporation
was used locally to introduce a transgene into
chick embryo and express the gene in spatial
and temporal manner (84, 85). By the micro-
electroporation technique, DNA molecules
were efficiently introduced into the optic ves-
icle (841, sensory placodes, surface ectoderm
(861, neuroepithelium of the CNS, and into the
somites and limb mesenchyme (87).
4 IMPROVING PLASMID D N A TRANSFER
MEDIATED BY GENE G U N
Gene gun is a physical way of administering
genes in vitro or in vivo. pDNA makes an elec-
trostatic complex with gold or tungsten micro-
particles. DNA-coated metal particles are
placed in the interior of Teflon-coated tubing,
and the DNA is readily expelled by a flow of
regulated, highly compressed helium gas. The
delivery is highly localized to the tissue part.
For regulating the speed and hence impact-
pressure, the projectiles can be targeted to dif-
ferent tissue depths and areas. The process
involves easy and speedy preparation of the
delivery vehicle while keeping DNA intact.
This technique sometimes allows DNA to gain
direct access to nucleus bypassing endosome/
lysosome wherein they would have possibly
enzymatically degraded. Because of the bene-
fit of accessibility, this technique is especially
suitable for gene transfer to skin (88) and for
superficial wounds (49,89). There are several
examples in various animal models that have
shown a high level of transgene expression in
the epidermis and dermis of the skin (90,91).
By the gene gun technique, mouse skin was
transfected with IL-6 and hemagglutinin-en-
coded DNA to elicit protective immune re-
sponses against equine influenza virus (92).
Several different large animals such as rhesus
monkeys (93), pigs (94), and horses (95) were
also immunized against virus by transfecting
gene encoding the viral antigen.
Other than skin, detectable transgene ac-
tivities were also noticed in various organs in-
cluding liver (96, 97), lungs (98), pancreas
658
(99), kidney (loo), muscle (101), and cornea
(102). Gene gun was also used to treat against
tumor growth (103). pDNA containing IL-12
gene delivered by gene gun to the epidermal
cells over an implanted intradermal tumor
gave detectable levels of the gene product at
the treatment site. This eventually led to com-
plete tumor regression within 7 days (104). A
particle bombardment of TGF p l encoded
plasmid to rat tissue enhanced the tensile
strength of the tissue by almost twofold com-
pared with the control (90). Porcine partial-
thickness wounds, when transfected with a
vector expressing epidermal growth factor
(EGF), showed an increased rate of reepithe-
lialization that duly shortened the healing
time by 20%, (105).
5 LIPID-BASED VECTORS
The lipid-based DNA delivery system was first
used on the premise that naked DNA on injec-
tion in vivo might be degraded by endogenous
DNAase. The lipidic delivery vehicle would
provide protection to the DNA during circula-
tion through the blood stream, and with nec-
essary chemical modification, this vehicle
would confer targeting potential to the DNA
towards specific cellular sites.
Lipids are a class of molecule that have a
tendency to self-assemble in aqueous or or-
ganic media. Depending on the molecular ar-
chitecture andlor the presence of co-solute,
they assume structures such as micelle, emul-
sion, or liposome in aqueous media and re-
verse micelle in organic media. Among them,
liposomes have shown wide diversity in biolog-
ical relevance. They constitute a bilayered
structure, which can encapsulate water-solu-
ble molecules in its aqueous hydrophilic core
or water-insoluble molecules in its hydropho-
bic bilayer. Liposome-based drug delivery sys-
tems have shown promise in clinical use, and
several products have already been approved
by FDA. Fraley et al. (106) have shown for the
first time that DNA could also be delivered to
cellular targets by encapsulating DNA in lipo-
soma1 aqueous core. They used a composite
liposomal system containing anionic and non-
ionic lipids but with a limited DNA encapsula-
tion efficiency. To enhance gene transfection
Plasmid DNA-Mediated Gene Therapy
and targeting ability of the liposome to specific
tissue site, Wang and Huang (107) used pH-
sensitive immunoliposomes made by coating
anionic Iiposomes with target specific anti-
body and encapsulating plasmid. These lipo-
somes, on intraperitoneal injection, showed
specific transfection to tumor cells in an as-
cites tumor model of nude mice. Very recently,
the immunoliposomes were revisited for tar-
geting brain. An exogenous gene was targeted
to the brain through the blood-brain barrier
by intravenous injection of pegylated immu-
noliposomes conjugated with antibody to rat
transferrin receptor. The whole entity was
targeted to the brain through the transcytosis
of transferrin receptor (108).
Felgner et al. (log), for the first time, used
cationic liposomes for gene transfection. The
primary idea was to electrostatically condense
DNA with more than one equivalent of cat-
ionic lipid and the negatively charged cells
would take up the net positively charged en-
tity when fed to cell. Since then, a plethora of
literature developed in fine tuning the chemi-
cal structure of lipids by systematic structure-
activity studies to elucidate the structure of
the optimal cationic lipid for gene transfec-
tion. Herein, pDNA played a significant role.
pDNA reconstructed with recombinant
markerlreporter gene and suitable promoter
is primarily used as a tool to screen for t'he
efficient transfection-lipid from a library of
cationic lipidic molecules. The electrostatic
lipid-DNA complex formed by first generation
cationic lipids is termed as "lipoplex." Cationic
polymers also condense DNA with or without
the presence of lipid; these polymers can de-
liver DNA to cells. This provides the basis for
the second-generation gene delivery systems.
Cationic polymers, cationic dendrimers, etc.
condense DNA to make a complex called
"polyplex." If liposomes, cationic or anionic,
are used to encapsulate the pre-condensed cat-
ionic polymer-DNA complex and are used to
deliver DNA to cells, then the whole entity is
termed as "lipopolyplex." We will talk about
these second-generation gene delivery sys-
tems later in this chapter.
5.1 First Generation of Cationic Lipids
Lipids with a glycerol backbone containing
aliphatic carbon chain(s): N-[1-(2,3-dioley-
5 Lipid-Based Vectors
10xy)propyll-N,N,N-trimethylammonium-
chloride (DOTMA), N-(2-hydroxyethy1)-N,N-
dimethyl-2,3-bis(tetradecy1oxy)-1-propana-
minium chloride (DMRIE), N-[I-(2,3-diole-
y1oxy)propyll-N-hydroxyethyl-N,N-dimethyl-
ammonium chloride (DORIE) (110), 2,3-di-
oleyloxy-N-[2(sperminecar-boxamido)ethyll-
N,N-dimethyl-1-propanaminiumtrifluoroac-
etate (DOSPA), N-(3-aminopropy1)-N,N-
dimethyl-2,3-bis(dodecy1oxy)-1-propanimi-
nium bromide (GAP-DLRIE) (1l l ) , N-[I-(2,3-
dioleoyloxy)propyll-N',Nr,Nr-trimethylam-
monium chloride (DOTAP) (112),and N, N42-
hydroxyethyll-N-methyl-2,3-bis(myristoyloxy)-
1-propanaminium iodide (DMDHP) (113).
These lipids are linked to glycerol moiety
through ether or ester-bond and have either a
single cationic charge with none, single, or
multiple hydroxyl groups or multiple cationic
charges by possessing spermidine derivatives
(Fig. 13.2).
Lipids with long chain alkylaminelalkylam-
ide moiety: dimethyldioctadecylammonium
bromide (DDAB) (114), DOGS (115), li-
popoly(~-lysine)(LPLL) (1161, GS2888 (117),
0,O'-ditetradecanolyl-N-(trimethylammonio
acety1)diethanolamine chloride (14DEA2)
(1181, diC14amidine (119), N,N-di-n-hexade-
cyl-N, N-dihydroxyethylammonium bromide
(DHDEAEi) (1201, and TFX. These are basi-
cally non-glycerol-based lipids, with single or
multiple charges. The charges arise because of
the presence of ammonium or multiple amines
or spermidines. Some of these lipids also pos-
sess multiple hydroxyl groups.
Lipids with cholesterol moiety: 3p-[N-(N',
Nr-dimethy1aminoethane)-carbamoyl] choles-
terol (DC-Chol) (121) and other cholesterol
derivatives (1221, biglycosylated cholic acid
derivative (123), and cholesterol with poly-
amines (124) and spermidines (125, 126).
Lipids with L-a-phosphatidylethanolamine
derivatives with spermidines: 1,2-dipalmitoyl-
phosphatidylethanolamidospermine(DPPES)
(116) or L-a-phosphatidylcholine with phos-
phonate diester (127, 128).
Lipids with imidazole derivative: 1-[2-[9-
(Z)-octadecenoyloxylethylll-2-[8l(Z)-heptade-
cenyll-3-[hydroxyethyl]imidazoliniumchloride
with long-chain alkylacyl -&tine ester (130).
There are many other lipids that have been
synthesized and tested for gene transfection.
Only a few of these lipids have shown consis-
tent and relatively high levels of transfection
in various cell lines and the in vivo system.
Most of these first-generation formulations do
not circulate long in the blood because the
DNAJcationic lipid complex disintegrates in
the presence of negatively charged blood pro-
teins. They are also grossly non-targetable to
cells. To overcome these problems, a number
of new delivery formulations have been cre-
ated and tested for gene
- transfection effi-
ciency. We will discuss these second-genera-
tion cationic formulations later in this
chapter.
5.2 Cellular Barriers for Transfection
To develop an efficient gene delivery system, it
seems necessary to understand the extra- and
intracellular processes involved in the overall
transfection mechanism. This will lead to un-
derstanding the mechanism, which is neces-
sary for developing novel lipid-based non-viral
vectors. For this purpose, cationic liposomes
and pDNA are used widely to understand the
cellular mechanism involved in the transfec-
tion (Fig. 13.3).
5.2.1 Structure of Lipid/DNA Complex.
Positively charged cationic liposomes electro-
statically interact with negatively charged nu-
cleic acid sequences to form fused, aggregated
complexes, which are capable of entering a
cell. These aggregates have a very heteroge-
neous distribution with respect to particle size
and net charge. The lipid-to-DNA mass and
charge ratio, which defines the size and net
surface charge of the complex, is an important
factor to determine transfection efficiency.
Hence, the structural features associated with
this kind of complex need to be interpreted.
Structural features revealed by electron mi-
croscopy include lipid-coated DNA strands,
aggregates of liposome intercalating DNA,
DNA entrapped between the lamellae of ag-
gregated multi-lamellar structure, and tubu-
lar structure consisting of fused liposome
around DNA (131-136). Polycations such as
polylysine or multivalent cationic lipid con-
dense DNA into small compact particles (137).

DOTAP
R={- R={A
R' = CH3, X = I R' = CH2C02CH3,X = Br
Phosphonate diester lipids
Figure 13.2. Structures of some representative cationic lipids.
Cells can efficiently uptake these particles
compared with the particles obtained by con-
densation of DNA by monovalent cationic lip-
ids (Sorgi et al., unpublished data).
5.2.2 Entry of DNA Into Cells. The pres-
ence of negatively charged proteins and glyco-
phospholipid impart negative charges on the
Plasmid DNA-Mediated Gene Therapy
L
DMDHP
R ={A
GAP-DLRIE
0
+ NH3
DOGS
cell surfaces. It was hypothesized that mem-
brane fusion between cationic liposome and
negatively charged cell membranes is the pri-
mary means of cell entry (109, 138). The hy-
pothesis was made based on the premise that
cationic and anionic liposomes readily fuse
(139, 140). It is conceptualized that the inter-
action of the cationic complex with the cell is
5 Lipid-Based Vectors

Nucleus

\\ A Inactive DNA /

Inactive DNA
/
Protein

0 Transport from
site of injection
*
00
0 u
Liposome DNA-liposome complex
Figure 13.3. Schematic illustration of the processes involved in gene delivery and expression.

primarily electrostatic and does not involve
any specific receptor for the cationic moiety.
However, Mounkes et al. (141) showed that
heparin sulfates, the highly anionic polysac-
charides, serve as an important cell surface
receptor for cationic lipid-DNA complexes.
Other experiments also suggested that the
mechanism of DNA-transfer to animal cells
from cationic liposomes might not entail a
simple fusion of liposomal and cellular mem-
branes (113). The non-differentiated edged
airway epithelial cells endocytose liposome-
DNA complexes consequent to their relatively
high negative charge and phagocytic activity
compared with differentiated cells (142). Evi-
dences now strongly suggest that slow endocy-
tosis of intact lipid/DNA complex is the pri-
mary method (143-146) for cellular entry.
5.2.3 Fate of Complex Inside the Cell: Es-
cape into Cytosol. After cellular entry, the
1ipidIDNA complex is engulfed into lower pH
662
compartment called "early endosomes" in the
peri-membranous region (147-150). In an at-
tempt to elucidate intracellular t r a c k i n g of
IipidIDNA complex, Zhou and Huang (117)
showed that liposome composed of lipopoly-
lysine (LPLL) and 1,2-dioeoyl-sn-glycero-3-
phosphatidylethanolamine (DOPE), condense
DNA to form electron dense particles recog-
nized by thin-section transmission EM, and
the majority of them reside in vesicular com-
partments. The endosomal contents usually
pass into the lysosome, the cell organelle in
the perinuclear region that houses a host of
degredative enzymes. The hydrolytic enzymes
degrade most if not all the lysosomal contents
(111,151-153). It becomes necessary that the
endosomal content must free itself at the en-
dosome stage to keep the DNA intact. Disrup-
tion of endosomal vesicle is visualized in the
liposomal system containing DOPE as a
helper or co-lipid. DOPE with its tendency to
promote significant polymorphic changes in
the lipid phase stimulates membrane fusion or
destabilization (117, 154, 155), which is fol-
lowed by leakage of endosomal content into
the cytoplasm. DOPE in aqueous media as-
sumes inverted hexagonal phase I1 structure,
which is frequently obtained in regions of
membrane where it fuses with another mem-
brane. Thus, one may assume that DOPE or
liposome formulation containing DOPE might
fuse with endosomal membrane and destabi-
lize it to leak out the content from the
endosome.
If the early endosomal release is not possi-
ble, another way to keep DNA intact in lyso-
some is to protect the DNA from lysosomal
degradation. Cationic liposomes formulated
with cholesterol believed to offer a useful role
in keeping DNAintact (121,156,157). Straub-
inger et al. (158) have demonstrated that the
lysosomal enzymes work at lower pH, i.e., pH
< 6. It was also shown that cholesterol-con-
taining liposomes, which possess greater sta-
bility and lower ion-permeability compared
with DOPE-containing liposomes, provide an
improved stability to the lipid-DNA complex
in the cytosol (158-160). It is easily conceiv-
able that if the endosomal content passes onto
lysosome before being released from endo-
somes, the lipidDNA complex could remain
secured in the lysosome.
Plasmid DNA-Mediated Gene Therapy
Cationic lipids can destabilize a cellular
membrane because of its intrinsic detergent
property. Therefore, destabilization of endo-
soma1 and/or lysosomal membrane may be a
contribution from the cationic lipids itself. In
the same context, it was shown that the cat-
ionic 1ipidDOPE or cationic lipid/cholesterol
liposome formulation exhibit surface anisotro-
pies in terms of increased liposomal surface
pH (161,162). The surface pH of the liposomal
formulations exhibits at least two pH units
higher than the pH of the solution a t which
they are made. Therefore, a liposomal solution
made at physiological pH may in reality ex-
hibit a surface pH r 9, which is detrimental
for both the stability of endosome and activity
of lysosomal enzymes. Endosomal disruptions
were also done with fusogenic peptides, which
promote pH-dependent fusion of small lipo-
somes when associated with lipid bilayer.
When these peptides were co-delivered with
lipidDNA complex, they imparted formidable
endosomal disruption by changing its usual
random coil conformation into amphipathic
a-helix conformation at lower pH, resulting in
consequent cytoplasmic delivery of DNA
(163).
5.2.4 Entry of DNA in Nucleus. The suc-
cess of lipid-mediated gene transfer is severely
limited by the inefficient transport of trahs-
fected DNA from the cytoplasm into the nu-
cleus (146). In general, macromolecules enter
the nucleus through nuclear pores. Molecular
aggregates with size more than 55 A in diam-
eter or molecular weight greater than 40 kD
use the nuclear pore complex (NPC) to access
nucleus. NPC bind with molecular aggregates
associated with nuclear localization signal
(NLS) peptide and help it to translocate across
the nuclear membrane (164-166). One of the
typical signals is the NLS from SV40 large T
antigen (PKRRRKV) (167), which had been
conjugated to many different molecules to
gain nuclear access (168, 169). NLS peptide-
conjugated plasmid DNA was delivered effi-
ciently inside the nucleus with an enhanced
gene expression (170). Similarly, SV40 T anti-
gen NLS co-delivered with DOTAP cationic
liposome-mediated efficient gene transfer and
expression in the cell (171). The genes deliv-
ered inside the nucleus require uncoating
6 In Vivo Use of Cationic LiposomeIDNA Complex (Lipoplex)
from the lipidic shell before the transcription
starts. It is generally assumed that pDNA is
displaced from the complex by anionic macro-
molecules in the nucleus. In this regard, an
alternative hypothesis for cytosolic release of
DNA from lipidDNA complex had been pro-
posed. Xu and Szoka (160) have demonstrated
by model studies that the pDNA was released
from cationic 1iposomeDNAcomplexes by an-
ionic liposomes exhibiting compositions mim-
icking the cytoplasmic face of the lipid mono-
layer of the cellular membrane. Membrane
destabilization followed by flip-flop of the lipo-
someDNA complex by the anionic lipids by
electrostatic interaction resulted in charge
neutralized lipidic ion pairs followed by re-
lease of pDNA.
To evade the nuclear transport, an alterna-
tive approach was developed that uses T7-
based cytoplasmic expression vectors. Here
the inefficiently nuclear-transported DNA
could be expressed in the cytoplasm itself
(172-175).
6 I N VlVO USE O F CATIONIC
LIPOSOME/DNA COMPLEX (LIPOPLEX)
6.1 Intravenous Injection
This is a widely used mode of gene delivery in
animals. On injecting liposome/DNA complex
(lipoplex) intravenously (IV), Stewart et al.
(156) for the first time showed the residence of
DNA primarily in heart and lungs even after 9
days with minimal toxicity. IV-injected lipo-
plex expressed transgene in almost all organs,
includingthe lung, kidney, heart, spleen, liver,
brain, etc., and the expression stayed for 9
weeks with apparently no treatment-related
toxicity (176). Toxicity and antitumor re-
sponse was evaluated in mice and pigs with
high doses of lipoplex containing MHC gene
incorporated pDNA (177).
Recently, more work has been done to in-
crease the overall transfection efficiency with
much higher targeting capability and repro-
ducibility of the liposomal delivery system
(159, 178, 179). Intravenously administered
lipidDNA complex avidly reacts with blood
components. So, it is necessary to keep the
complex intact in the blood until it reaches the
organ of interest. Sakurai et al. (180) have
663
shown that in the presence of erythrocytes,
cationic lipid/cholesterol formulation didn't
induce fusion between erythrocytes, whereas
the cationic 1ipidDOPE formulation possess-
ing high fluidity in its structure induced fusion
between the erythrocytes after a short incuba-
tion period. This offered an explanation for
why cholesterol makes more superior formu-
lation with cationic lipids for in vivo purposes
(159).
A repeated systemic intravenous injection
of cytokine gene (IFN pl) by lipoplexes gave a
systemic expression of human interferon+ in
mice, thereby increasing the possibility of cy-
tokines used for therapeutic purposes in a sys-
temic manner (181). Hwang et al. observed an
enhanced and highly selective liver targeting
by IV injecting cationic 1iposomeIpDNA con-
taining p-sitosterol P-D-glucoside (Sit-G)
(182). IV injections showed gene expression in
all major organs including the heart, lung,
liver, spleen, and kidney, with the lung being
most efficiently transfected (178, 183). For ef-
ficient targeting and gene expression in the
lung, intravenous injection was favored over
intratracheal instillation. Uyechi et al. (184)
have shown by injecting fluorescently tagged
lipoplexes through the vein that the entire
lung lobe was homogenously fluorescent,
whereas intratracheal administration re- -
sulted in regional distribution of lipoplex, con-
centrated around bronchiales and distal air-
ways.
6.2 Direct injection
Direct injection to tissue is a common ap-
proach for cationic lipid-mediated gene ther-
apy. Intratumoral injection of DNA-liposome
complexes containing either E6 or E7 anti-
sense plasmid resulted in significant growth
inhibition of C3 tumors grown in a syngeneic
mouse model (185). E6 and E7 are two onco-
genes responsible for the maintenance of the
malignant state of HPV-position tumors. Na-
be1 et al. (186) have directly injected recombi-
nant pDNA containing murine class I major
histocompatibility (MHC) gene into localized
arterial segment of various major organs and
showed that the direct gene transfer by liposo-
ma1 transfection did not lead to treatment re-
lated toxicity, autoimmunity, or gonadal local-
ization of the transgene in mice. The toxicity

of gene delivery by DNA-liposomes was also
analyzed in pigs and rabbits in vivo. There
were no clinically significant immunopathol-
ogy in major organs such as the brain, heart,
lung, liver, kidney, spleen, and skeletal mus-
cles. To induce local tumor immunity in pa-
tients with stage IV melanoma, Nabel et al.
(187)injected pDNA encoding MHC class I an-
tigen complexed with cationic lipid directly
into the cutaneous tumor nodules. Treated le-
sions exhibited presence of T-cells, followed by
an enhanced reactivity of tumor infiltrating
lymphocytes. As a result, local inhibition of
tumor followed by complete diminution of tu-
mor was observed in some of the treated pa-
tients. Mohr et al. (188) have shown that di-
rect liposome-pDNA complex injection to
intrahepatic hepatocellular carcinoma pro-
duced by human HCC cells seemed far supe-
rior to systemic administration for gene ther-
apy for localized intrahepatic tumors, because
the direct administration to tumors left the
surrounding normal hepatic cells untouched.
In another typical example, in vivo, direct in-
tratumor injection of plasmid containing the
coding sequence for the human IL-2 gene com-
plexed with cationic lipid formulation resulted
in retention of intact pDNA in the tumor tis-
sue and IL-2 secretion by cell cultures derived
from the injected tumors. Formulation of this
lipid with the cationic lipid inhibited DNA
degradation and enhanced in vivo transfection
efficiency over pDNA alone (189).
Airway administration of liposome com-
plexes was used for the treatment of pulmo-
nary diseases including cystic fibrosis. Cat-
ionic liposome/DNA complex showed no
adverse effect towards airway epithelial integ-
rity (190); therefore, the cationic lipid-based
delivery system proved to be appropriate for
use in human trials for cystic fibrosis (CF). A
series of me-clinical trials were done in CF
patients with intranasal instillation to evalu-
ate the risk factors associated with the treat-
ment (191-193). Because there was no appar-
ent toxicity associated with lipoplexes as was
seen from these trials, progress had been
made in delivering the complexes to the entire
lung by aerosol in CF patients (192,194,195).
By nebulization, the DNA-liposome complex
was delivered into the airways of mutant mice
to obtain human cystic fibrosis transmem-
Plasmid DNA-Mediated Gene Therapy
brane conductance regulator (CFTR) cDNA
expression in the respiratory tract (196). A
study conducted on CF patients revealed that
pCMV-CFTRJcationic liposome complex on
administration to the nasal epithelium gave
no evidence for excess nasal inflammation, or
any adverse events related to active treat-
ment. Gene transfer and expression assayed
by PCR revealed the presence of transgene
DNA in seven of the eight treated patients up
to 28 days after treatment (192). Intranasal
instillation technique was also used in the
mouse model, to incorporate cationic lipid/
DNA complexes (112, 197). The CFTR dys-
functional gene gives rise to multiple defects
in airway epithelia such as altered C1- and
Na+ permeability. Zhang et al. (198) have
shown that the goblet cells were more effi-
ciently targeted with lipoplexes than any
other cells in the entire spectrum of lung air-
way epithelia. This was ascertained by the fact
that an efficiently reduced mucous sulfation to
levels seen in non-CF airways was observed
with lipoplex/DNA despite low levels of CFTR
gene expression in lung epithelial cells in hu-
man bronchial xenograft model of mice com-
pared with non-CF airways of control mice.
This kind of apparent complexities in CFTR
function presented challenges in the design of
different lipoplex formulations that were ca-
pable of generating the endogenous pattefns
of CFTR gene expression in specific lung epi-
thelial cells.
6.3 lntraperitoneal Injection
Intraperitoneal (IP) injection of DNA/lipoplex
was done to transfect cells in the peritoneum
region. Nude mice bearing disseminated hu-
man ovarian tumors derived from p185-over-
expressing SKOV-3 ovarian cancer cells were
injected with E1A genellipoplex intraperitone-
ally (199). These tumors resemble stage I11
human ovarian cancer. The expression of E lA
protein decreased the expression of pl85 onco-
protein and hence increased the survival rate
of mice (200). 70% of the treated group sur-
vived for 1.5 years from the last injection, but
the untreated group barely survived more
than 16 weeks. The treatment of complex con-
taining 1/13 of the original lipid dose also
worked as efficiently as the normal dose.
There was no apparent toxicity or major organ
7 Second Generation of Lipidic Delivery System (Lipopolyplex)
pathologic change. There was no trace of E1A
DNA in the liver, lung, heart, spleen, brain,
uterus, and ovaries of the treated mice even
after 1.5 years (199).
7 SECOND GENERATION OF LlPlDlC
DELIVERY SYSTEM (LIPOPOLYPLEX)
7.1 Polycation-Condensed D N A Entrapped
in Cationic Liposome: LPD-I Formulation
One of the prerequisites for a bio-entity enter-
ing the cell and nucleus is to possess a small
size. The delivery of DNA was efficient
through cationic lipidic formulation-mediated
gene delivery, but the nuclear transport from
cytoplasm was quiet an inefficient process
(146). Generally, lipoplexes formed by multi-
ple charged cationic lipids made small ( ~ 2 0 in different cell lines. The extent of transfec-
nm), highly condensed DNWipidic complex.
These complexes were found to be more trans-
fecting than the lipoplex formed by mono-cat-
ionic lipids and DNA. Synthetic cationic poly-
mers like poly-L-lysine (PLL) could also
condense DNA into very small compacted par-
ticles. This compact complex was allowed to
freely mix with cationic liposomal solution.
The overall complex formed was termed
LPD-I (200). A sucrose gradient ultracentrif-
ugation of the heterogeneous mixture showed
the existence of various populations associ-
ated with varied amount of lipid. Negative-
stain EM studies of purified complexes showed
electron dense structures ranging from elon-
gated rod-shaped to ball-shaped particles. The
purified fractions were severalfold more trans-
fection efficient than unpurified ones. The
fractions, which contained more lipids, were
more efficient in gene transfection than those
containing fewer lipids. The transfection effi-
ciency of LPD-I was severalfold higher than
the corresponding cationic liposome-DNA
complex. It is hypothesized that the small
compacted structure of DNA resulted in high
cellular and nuclear uptake, the DNA is pro-
tected efficiently against the enzymatic degra-
dation, and PLL may have mimicked the nu-
clear localization signal for nuclear delivery of was rapidly removed from circulation by the
DNA. An alternative cationic polymer, prota-
mine sulfate, from salmon sperm was used as a
substitution for PLL (201). Protamine sulfate
is a small (MW ~ 4 0 0 0versus -18,500 for
665
PLL), highly positively charged peptide and
very basic because of the presence of 21 argi-
nine residues, which contains a nuclear local-
ization signal (202). It is naturally occurring
(whereas PLL is synthetic), a United States
Pharmacopeia (USP) grade compound, FDA-
approved, and finds its use as a n antidote to
heparin-induced anti-coagulation. With a rou-
tine history of human administration, the is-
sues of toxicity and immunogenicity were min-
imal for protamine sulfate.
Both protamine sulfate and PLL were com-
pared for their transfection efficiencies. With
the same amount of DNA and cationic lipid,
PLL reached its efficiency plateau at an
amount one-half the amount of motamine sul-
fate, but the overall efficiency of protamine
sulfate was two- to sevenfold higher than PLL
tion efficiency for LPD-I was 7- to 45-fold
higher than the levels shown by DNNcationic
lipid complex. Phosphate and free base form of
protamine showed lower levels of transfection
and the activity was comparable with lipoplex.
It is noteworthy that arginine, which exists as
a salt of sulfate, and the lysine as free base or
as a phosphated salt, showed a variance in the
relative transfection efficiencies even though
the percentage of basic (arginine + lysine) res-
idues remained relatively constant.
On intravenous injection, LPD-I made with
DOTAP had shown very high gene expression
in the heart, lung, liver, spleen, and kidney,
with the highest expression found in the lung
(178). The in viuo gene expression had steadily
increased with increase in cationic lipid andlor
protamine, but the protamine-DNA charge ra-
tio was kept below 2:l to avoid forming large
aggregates. After LPD injection transgene ex-
pression was detected as early as 1 h, it peaked
at 6 h and declined thereafter. The lung con-
sistently showed the highest gene expression,
which lasted for 2 days. Even after 4 days of
LPD injection, gene expression could still be
detected in the lung, spleen, and liver.
In a bio-distribution study with labeled
DNA and liposome, it was revealed that DNA
liver after injection of protamine-DNA com-
plexes and uptake by the lung was always less
than 10% of injected dose at all time points.
However, DNA formulated in LPD was

trapped in the lung to an extent of 40% within
5 min of LPD injection. Southern blot analysis
revealed that the pDNA injected with LPD
was detectable in the lung in much higher
quantity than the control even 6 h after IV
injection. Intraportal injection gave signifi-
cantly lower gene expression than with intra-
venous injection.
7.2 Polycation Condensed DNA in Anionic
Liposomes: LPD-II Formulation
Some disadvantages always accompany the
cationic lipid-mediated gene transfection. Cy-
totoxic effect in cells/tissues is the primary
concern, which requires early attention. The
cationic lipids exhibit relatively large sizes,
whereas complexation with DNA provide sub-
optimal DNA condensation and have limited
efficiency and lack of tissue specificity. Anionic
liposomes used in gene therapy have shown
poor encapsulation efficiencies because of
large size and excessive negative charge of un-
condensed pDNA. The use of anionic lipo-
somes was revisited during a recent approach,
which also used the concept of condensing
DNA by cationic polymers. A delivery vector
was developed wherein polylysine-condensed
pDNA was trapped in folate-targeted anionic
liposomes through charge interaction. It had a
structural similarity to LPD-I and it was
named LPD-II(203). It differed from LPD-I in
that anionic lipids instead of cationic lipids
were used. This novel vector was more effi-
cient and less cytotoxic compared with con-
ventional cationic liposomal vectors.
Folate-targeted LPD-I1 particles were gen-
erated by mixing anionic liposomes composed
of DOPE/cholesterylhemisuccinate (CHEMS)/
folate-polyethyleneglycol (PEG)-DOPE and
the cationic DNA-polylysine (1:0.75, w/w)
complexes. Structural analysis of LPD-I1 by
negative-stain EM showed that the DNA-poly-
lysine (which appears individually as rod
shaped) and lipid complex seemed to be a
highly electron dense, spherical core with a
low-density coating. The mean diameter of
these particles was 74 2 14 nm, i.e., smaller
than the empty liposomes.
KB cells expressing folate receptors were
transfected with LPD-I1 particles containing
luciferase reporter gene, and high transfection
efficiency was observed. The activity could be
Plasmid DNA-Mediated Gene Therapy
inhibited by the presence of excess free folate.
Control LPD-I1 particles generated with non-
targeted liposomes was only active at low lipid1
DNA ratios, suggesting that the transfection
by LPD-I1 particles was only receptor depen-
dent when the over all charge was negative.
Compared with DC-Chol/DOPE/DNA lipo-
some complexes, LPD-I1 showed -20- to 30-
fold more transfection activity. On replacing
DOPE with DOPC in the original formulation,
the transfection was severely reduced. This in-
dicated that the fusogenic activity of DOPE
was essential for the transfection activity of
LPD-I1 particles.
The therapeutic applications of antisense
oligonucleotides (ODN) are currently limited
by their low physiological stability, inefficient
cellular uptake, and the lack of tissue specific-
ity. The use of various vectors renders phos-
phodiester (PO) ODN resistant to enzymatic
digestion. KB cells, which overexpress folate
binding protein, were also transfected with
LPD-11-containing targeting ligand folate to
deliver ODN against epidermal growth factor
receptor (EGFR). This resulted in down-regu-
lation of EGFR and growth inhibition of KB
cells (178). The modified backbone ODN, i.e.,
phosphorothioate (PS) and monomethylphos-
phonate (MP), are more stable to enzymatic
degradation compared with PO ODN, but they
suffer from increased toxicity and decreased
specificity. In one study, PO ODN against
EGFR had shown growth inhibitory effect to
KB cells compared with that of PS/PO ODN
when delivered with LPD-11, indicating that
LPD-I1 could also protect PO ODN from the
attack by enzymes inside cells (204).
8 EMULSION-MEDIATED GENE TRANSFER
8.1 General Development
Emulsions are one of the most widely studied
colloidal dispersion systems for the delivery of
drugs (205,206). The oil-in-water emulsion is
made of oil dispersed in an aqueous phase with
a suitable emulsifier such as phospholipids
and non-ionic or ionic surfactants. Castor oil
or soybean oil is predominantly used as the
core oil phase. Non-ionic surfactants such as
Tween, Span, Brij, and pluronic copolymers
are used as co-emulsifiers. The ionic co-emul-
9 Gene Delivery by Polymeric Systems (Polyplex)
sifiers are phospholipids or cationic lipids. A
number of structure-activity studies had been
done with different emulsion formulations,
which were subsequently used for gene deliv-
ery in uitro and in uiuo. The non-ionic surfac-
tants such as Tween, Span, Brij, and pluronic
copolymers were found to be excellent co-
emulsifiers when used along with castor oil,
DC-Chol, and DOPE (207). The in vitro trans-
fection study on BL-6 cells showed that the
Tween surfactant-containing formulations
had more serum resistivity and exhibited
higher transfection in serum-containing me-
dia than in the absence of serum. One of the
Brij-containing formulations, i.e., one with
2-oxyethylene chains, showed the highest
transfection efficiency in presence of serum.
The toxicity of each formulation is minimal. In
DOTAP, soybean oil, and pDNA emulsion
complexes, it was observed that despite the
change of 6 potential with the varying amount
of DNA, the structure and the size of the emul-
sion complex remained mostly unchanged
(208). The stability of this emulsion complex
was shown to be high and inhibited DNAase-I
digestion of pDNA. In serum-containing
media, the emulsion showed much higher
transfection efficiency compared with the lipo-
fectamine/DNA transfection complex. Inclu-
sion of polyethyleneglycol-PE in the emulsion
complex created a high-level transfection that
was observed even in the media containing
90% of serum. This result suggested that in
uiuo transfection could be done with this emul-
sion complex. Another soybean oil-DOTAP
emulsion was used to transfer genes to the epi-
thelial cells of the mouse nasal cavity through
intranasal instillation. The emulsion showed en-
hanced stability against heparin exchange and
exhibited higher level of transfection compared
with commercially available liposome/DNA
complexes in nasal cavity mucosa (209).
8.2 Reconstituted Chylomicron Remnants
for Gene Transfer
Chylomicrons are triglyceride-rich lipopro-
teins that are slowly modified during the cir-
culation in blood. The core glyceride structure
is hydrolyzed by the lipoprotein lipase. It is by
the apolipoprotein-specific receptors that the
hepatocytes in the lever consume these rem-
nants (210,211). It had been shown that even
reconstituted chylomicron remnants formed
by purified lipids could be taken up by the
hepatocytes following intravenous injections
(212). In our laboratory, we modified the sys-
tem and used it for delivery of DNA into liver
cells (213). DNA is predominantly hydrophilic
in nature and therefore cannot be included in
the hydrophobic interior of the reconstituted
chylomicron remnants (RCR). 3P-[N-(N',
N' ,N1-Trimethylaminoethane)carbamoyl]
cholesterol (TC-Chol), a quaternary ammo-
nium analog of DC-Chol, was used in various
amounts to make complexes with DNA. The
resultant hydrophobic complexes extracted
from aqueous solution were emulsified with
commercially available lipids (olive oil: L-a-
phosphatidyl choline, L-a-lysophosphatidyl
choline, cholesteryl oleate) by homogeniza-
tion. DNA/TC-Chol complex was incorporated
into the internal oil space of RCR and re-
mained protected against DNAase I digestion.
The RCR-containing DNNTC-Chol complex
showed transgene luciferase expression in the
liver 100-fold higher than with naked DNA
injection when injected intraportally. The ex-
pression was also seen in the spleen, lung, and
heart, but was 25- to 800-fold lower than in the
liver. The gene expressions obtained through
tail vein injection were 100-fold lower than of
the mice injected intraportally. This was likely '
because the RCR didn't contain any apoli-
poproteins on its surface, which could have
otherwise facilitated the receptor-mediated
uptake. Moreover, the colloidal stability might
not be as good as the ones containing apoli-
poproteins. RCR didn't employ any protein or
peptide that acts as an antigen. So, it could be
injected repetitively by using a catheter
method that had been established for multiple
portal vein infusion (214). We have recently
improved the formulation by adding pegylated
lipid to the surface of RCR and showed that
the circulation time of the particle was pro-
longed significantly (215).
9 GENE DELIVERY BY POLYMERIC
SYSTEMS (POLYPLEX)
9.1 General Development and Uses
An alternative non-viral gene delivery system
has been developed that uses polymers, either
668
cationic or neutral. Cationic polymers are pre-
dominantly used because they efficiently con-
dense DNA to very small particles. These com-
plexes are called "polyplexes." Wu et al. (216)
used polylysine-asialo-orosomucoidconjugate
to condense pDNA and target the pDNA to
liver. pDNA condensed with protamine/poly-
lysine-conjugate of iron transport protein,
transferrin, was efficiently delivered to eu-
karyotic cells (217). pDNA condensed by CD3
antibody-polylysine conjugate showed recep-
tor-mediated endocytosis to efficiently inter-
nalize pDNA into T-lymphocytes (218). De-
pending on the cell-binding ligand, specific
targeting was obtained in different cell lines
(219).
Several of the most effective polymeric de-
livery systems were polyamidoamine den-
drimers (220) and polyethyleneimines (PEI)
(221). Being non-biodegradable, these syn-
thetic polymers posed a potential toxicity to
cell; therefore, biodegradable polypeptides
like PLL and protamine were used for conden-
sation and delivery of gene, but they had lim-
ited efficacy in transfection (222, 223). They
were usually used with cationic lipids to ob-
tain enhanced transfection activity (178,201).
Among the biodegradable polymers, chitosan
(224) and P-cyclodextrin-based polymers
(225) were also used for gene transfection.
Although the cationic polymers shared the
same mechanism of DNA condensation, the
transfection efficiency greatly varied between
polymers. Even different molecular weights
and isomeric forms of the same polymer
showed different physicochemical characteris-
tics, transfection efficiency, and toxicity (221,
226-228).
Ligand-conjugated polymers were used for
in vivo targeting and expression of genes. Kir-
cheis et al. (229) reported an enhanced level of
transfection in subcutaneous neuro2a tumors
on intratumoral injection of transferrin-PEI/
pDNA compared with naked pDNA injection.
On pegylating the Tf-PEIIpDNA complex, the
whole entity became serum resistant without
losing its targetability, and the complex could
be efficiently targeted to neuro2a tumors in a
mouse model after IV injection (230). Alvarez-
Maya et al. (231) cross-linked neurotensin
with PLL and made polyplexes with pDNA.
On injecting polyplex into the substantia nigra
Plasmid DNA-Mediated Gene Therapy
of rat, a high level of transfection was ob-
served in nigrostriatal dopamine neurons and
was detectable up to 15 days.
EBV-based plasmid vector containing thy-
midine kinase (TK) gene coupled with poly-
amidoamine (PAMAM)dendrimer (EBVIpoly-
plex) was used in suicide gene therapy of
cancer (232). Huh7 hepatocellular carcinoma
(HCC) tumors in SCID mice were injected
intratumorally with TK genes containing
EBV/dendrimer polyplex to show remarkable
suppression of tumor growth, leading to pro-
longation of survival time. Gene transfer to
the lung was obtained by intravenous injec-
tion of G9 PAMAM dendrimer-complexed
pDNA into mice. This resulted in high levels of
transgene expression in the alveoli at 12 and
24 h, followed by a second peak of expression
3-5 days after administration. However, the
direct endobronchial administration of this
polyplex primarily targeted bronchial epithe-
lium (233).Topical in vivo delivery of pDNA to
the hairless mice skin was done with PAMAM
dendrimer polyplex. The polyplex was incor-
porated in or coated on the surface of PO~Y(DL-
lactide-co-glycolide) (PLGA) or collagen-based
biodegradable membranes (234).
In an effort to transfer genes to rabbit ca-
rotid artery, Turunen et al. (235) used DNA/
fractured dendrimer (generation 6) polyplex
to obtain a high level of gene transfer (4.4%)
compared with what was obtained by lipoplex.
The arterial gene transfer was particularly
useful because it could be used as a tool for
treating various vascular diseases. In vivo
gene transfer methods in this study employed
a gene delivery reservoir (collar) around the
carotid artery, which served as a reservoir for
the gene delivery solution. This type of local
gene transfer with cationic polymer-pDNA
complexes provided a technically efficient way
of treating arterial diseases during vascular
surgeries. These dendrimer-based polyplexes
showed a clear advantage over polylysine in
that they can buffer the drop of endosomal pH
inside the endosome leading to an increase in
transfection efficiency (236, 237, 223).
Qin et al. (238) used starburst PAMAM
dendrimers to transfer genes into a murine
cardiac transplantation model. These star-
burst dendrimers are a special class of its kind,
which are highly branched spherical polymers
10 Non-Viral Vector-Related Cytotoxicity
with large number of amino groups on the sur-
face. At the time of transplantation of whole
heart in the recipient mice, the dendrimerlp-
gal pDNA was directly injected into the graft
tissue. X-gal staining revealed a highly effi-
cient and wide spread transgene expression in
both myocytes and the graft infiltrating cells
with the peak lasting up to 14 days. For organ-
transplantation, severe tissue rejection is a
common immune response. Viral IL-10, a cy-
tokine synthesis inhibitory factor, is able to
regulate a variety of negative immune re-
sponses by suppressing the synthesis of IFN- y
or inhibiting IL-1, IL-6, IL-8, IL-12, and
TNF-a! (239-242). Direct injection of this den-
drimerlviral IL-10 gene with a-MHC promoter
polyplex to the cardiac tissue showed an in-
creased survival of the cardiac allograft. As
little as 0.31 pg of the injected pDNA led to an
increased mean survival from 13.9 to 38.6
days.
9.2 Targeted Gene Delivery by Antibodies
Conjugated with Polycations
Polyamines had shown efficient transfection
to lung endothelium. An efficient targeted
transfection vector to the lungs could be
achieved by conjugating a targeting ligand
against platelet endothelial cell adhesion mol-
ecule-l (PECAM-l) to polyamines. This li-
gand-polyamine complex was targeted effi-
ciently to the pulmonary endothelial cells. A
chemical vector was synthesized by covalent
conjugation of polyethylenimine (PEI) and
anti-PECAM antibody (Ab) (243). The cat-
ionic complex was shown to deliver DNA spe-
cifically to mouse lung endothelial cells. The
highest gene expression was obtained at rela-
tively low plus-to-minus charge ratios. The
PEI conjugated with a control IgG did not en-
hance transfection of mouse lung endothelial
cells. Intravenous injection of this anti-PE-
CAM Ab-PEI /DNA showed an increased lung
expression in mice compared with other
modes of injection (243).
TNF-(win blood was found to be about five-
fold less in the mice injected with PEI-anti-
PECAM AbDNA than in the mice injected
with PEIDNA. The decrease in TNF-a was
partially reversible by pretreating mice with
Ab to PECAM. The immunosuppressant dexa-
methasone not only improved the persistence
and level of gene expression in the lung but
also shortened the refractory period for re-
peated dosing when injected along with anti-
PECAM AbIDNA. This supported a potential
therapeutic role of dexamethasone in lung
gene transfer with Ab-polymer conjugates.
Similarly, Ferkol et al. (244) targeted the
polymeric immunoglobulin receptor (pIgR),
which is expressed in lung and liver tissues,
and transferred pDNA to these tissues. The
targeting ligand, anti-secretory component
(SC) Fab antibody, was covalently linked to
poly-L-lysine. The polycation, after condens-
ing pDNA, was delivered successfully to air-
way epithelium in uiuo. Tissues that do not
express the pIgR, the spleen and heart, were
not transfected. In addition, conjugate pre-
pared with irrelevant Fab fragments did not
produce detectable transgene activity. This
complex specifically targeted pIgR-bearing tis-
sues, but after repeated dosing, increased hu-
moral immune response against anti-SC Fab
antibody was observed (245).
10 NON-VIRAL VECTOR-RELATED
CYTOTOXICITY
It is known from the 1980s that bacterial DNA
stimulates the formation of cytotoxic IFN-a!,
p, and IL-12 when the DNA is taken up by
macrophages. It in turn leads to NK cell acti-
vation and production of pro-inflammatory cy-
tokine IFN-y. This is accompanied by the
proliferation of B-cells and therefore the re-
duction of apoptosis and release of IL-6 and
IL-12 (246-249). These pro-inflammatory ef-
fects were found to be caused by some im-
muno-stimulatory sequences in prokaryotic
DNA that contained unmethylated CpG dinu-
cleotide motif flanked by two 5' purines and
two 3' pyrimidines. Plasmid DNA, which is
derived from bacterial DNA, induces these im-
mune responses. The unmethylated CpG mo-
tif-containing sequence occurs four times
more frequently in prokaryote DNA than in
eukaryotic DNA. Moreover, the CpG motifs
are usually 75% more methylated in mamma-
lian DNA than in prokaryotic DNA (250,251).
On methylation of the cytosine bases in plas-
mids, the immuno-stimulatory effect is de-
creased considerably (252). Immature den-
670
dritic cells tend to produce pro-inflammatory
IFN-a, P, IL-6,Il-12, and TNF-a during expo-
sure to CpG containing oligonucleotide or bac-
terial plasmid (253,254).
These immune-stirnulatory effects leading
to inflammatory cytokine productions had a
negative impact on the systemic gene delivery
of caionic lipid/DNA complexes. On recogni-
tion of pDNA by splenic macrophages during
circulation, there was every chance that the
pDNA would elicit an immune response,
which might lead to phagocytosis of the com-
plex and hence decreased transgene expres-
sion. High levels of pro-inflammatory cytokine
also led to inactivation of several promoters,
resulting in a decrease in transgene expres-
sion (255-257). The death of animals by high
dose lipoplex injection for obtaining high
transgene expression (258) could be attrib-
uted both to the high concentration of lipid
and pDNA mediated toxicity. In the case of
local lipoplex administration in animals, min-
imal toxicity was observed. However, on intra-
venous injection or intratracheal instillation,
high levels of IFN-7and TNF-a were observed
(259-261). Minimized CpCr triggered inflam-
mation and immune responses followed by
prolonged gene expression were obtained by
pretreating cytokine-neutralizing antibodies
during intravenous injection of lipoplex (259).
Repeated dosing without any antibody treat-
ment led to silenced transgene expression af-
ter 1 or 2 weeks (178,259).
It is not clear how cytokine production de-
creases the transgene expression, but various
efforts to minimize CDG-related immune re-
*
sponses and toxicity and to enhance transgene
expression is worth mentioning. Hofmann et
al. (262) have used PCR amplified fragments
containing encoded therapeutic gene and reg-
ulatory elements for preparing LPD. On deliv-
ering, a similar level of gene expression com-
parable with pDNA lipoplex was obtained.
However. much lower level of cvtokine
" re-
sponse was observed, which sustained the
gene expression for a longer period than
pDNA. PCR fragments contain fewer CpG mo-
tifs than the full-length pDNA, this led to re-
duced CpG-triggered adverse effects. Yew et
al. (263) have shown that during mutating
CpG or its flanking motifs in the plasmid, a
decreased level of cytokine and increased
Plasmid DNA-Mediated Gene Therapy
transgene expression could be obtained. A lim-
ited interaction of plasmid with immune cells
could also lead to decreased cytokine response.
It could be achieved by sequentially injecting
cationic liposomes and free pDNA. Song et al.
(264) used this process for the purpose of effi-
ciently transfecting the lung by prolonging the
residency time and interaction of DNA with
pulmonary endothelium. Tan et al. (255) used
the same concept to show that the sequential
injection led to the formation of a lower level of
cytokines compared with lipoplex injection. It
is evident from the above efforts that under-
standing the detailed mechanism of CpG-in-
duced immune response is required for in-
creasing the efficacy of pDNA-mediated gene
delivery.
11 CONCLUSION
Since the first attempt of using plasmid DNA
for gene delivery to cells, two decades have
seen many attempts to enhance the efficacy of
genetic vectors, to test various therapeutically
important genes, and to understand the mech-
anism of the gene delivery, associated toxicity,
and factors inhibiting gene expression. A
number of gene therapy clinical trials have
also been performed. Although no single vec-
tor is superior over other vectors, each in vivo
gene transfer application will find its vector
system for optimal performance. Understand-
ing cellular barriers and possible means to
overcome will undoubtly further improve the
performance of a non-viral vector.
12 ACKNOWLEDGMENTS
The original work from our lab described
in this review has been supported by NIH
Grants CA-74918, AR-45925, DK-44935, and
AI-48851. Discussions with Dr. Song Li are
gratefully acknowledged.
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Index
Terms that begin with numbers
are indexed as if the number were
spelled out; e.g., "3D models" is
located as if it were spelled
"ThreeD models."
A-23187,214
A-63162,213
A-64077,213
A-79175,214
A-79176,214
A-787733,214
Absorption, distribution, metab-
olism, and excretion
(ADME)
biguanides, 21-23
a-glucosidase inhibitors, 31-33
insulin and analogs, 5-8
insulinotropic agents, 15-17
thiazolidinediones, 24
ABT-080,219
ABT-761,214-215
ABT-963,240
Acarbose, 31,32,33,34-36,36
Accolate, 225-226
Accutane, 320,373
Acetohexamide, 12,16
Acetrizoate, 497
angiography application, 569
histamine release, 558
neurotoxicity, 555
particulate, 576
pharmacokinetics, 561
properties, 504
protein binding, 556, 557
urography application, 571
Acetyl CoA carboxylase
biotin dependent, 404
N-Acetyltransferase-2
single nucleotide polymor-
phism~,630
Acitretin, 320,373374
Acne
retinoids used for, 372-373
Actos, 25
Acute coronary syndrome
single gene pharmacogenetic
studies in genes influencing
progression, 638-639
Acyclovir
classification in various coun-
tries, 430
Adapalene, 320,373
Adaptive toxicological responses
to UV radiation, 464-465
Adenine, 619
Adequate Intake, 368
biotin, 405
folic acid, 411
niacin, 397
pantothenic acid, 402
vitamin A, 371
vitamin B,, 389
vitamin B,, 392
vitamin B,, 400
vitamin C, 418
vitamin D, 379
vitamin E, 384
vitamin K, 387
ADME studies. See Absorption,
distribution, metabolism,
and excretion (ADME)
Adrenalin
role in insulin regulation, 2
a-Adrenergic receptor
gene expresion profiling study,
612
0,-Adrenergic receptor
single nucleotide polymor-
phism~,635-636
0,-Adrenergic receptor
single nucleotide polymor-
phism~,636
P-Adrenergic receptor antago-
nists. See 0-Blockers
Advanced glycation end-prod-
ucts (AGES),4
Advance notice of proposed rule-
making, 425
Adverse drug events
defined, 545-546
reporting, 473-474
Adverse drug reactions
defined, 546
Albumin. See Serum albumin
Albuterol
impact of polymorphisms on
effectiveness, 636
679
Aldose reductase
in diabetes, 3-4
Alitretinoin, 320,374
Allele-specific amplification,
624
Alopecia, 433. See also Hair
growth disorder drugs
Alosteron
removed/restricted by FDA,
642
Alprostadil, 299301,442
adverse effects, 443
development of, 448
mechanism of action, 446
pharmacokinetics, 444
structure-activity relationship,
447
Alternative macrophage activa-
tion-associated CC chemo-
kine (AMAC),150
Alzheimer's disease
single gene pharmacogenetic
studies in genes influencing
progression, 638-639
Amaryl, 13
AMD2763,168
AMD3100,168-170
AMD3329,168
AM1 25
radiopaque material, 491
AM1 227
radiopaque material, 491
Amidotrizoate
neurotoxicity, 554
Amino acids
novel chi-constrained, 52
Arninobenzoate sunscreens, 460,
461
absorption and disposition,
462-463
development of, 470
structure-activity relationship,
467-468
Amiodarone
P-glycoprotein inhibitor, 631
P-glycoprotein substrate,
631
Amitriptyline
CYP2D6 substrate, 627
 Index

Amlodipine Apolipropotein E Basophils

CYP3A4/5/7 substrate, 627 impact of polymorphisms on chemokinelcytokine receptors,
AMP-activated protein kinase Alzheimer's, 637, 638-639 133
system Apomorphine, 450 Bayer ~7195,222- 223
role in carbohydrate metabo- Arachidonic acid Bay X1005,217-219
lism, 23 leukotriene biosynthesis from, Bay Y1015,218
a-Amylases, 33 205-207 B-cells
Amylopectin, 33 Arginine vasopressin, 67 chemokine/cytokine receptors,
Amylose, 33 Arteriography, 567. See also Ra- 133
Anagen phase, of hair growth, diopaques Benzophenone sunscreens
425 CT application, 568 structure-activity relationship,
Anakinra, 174 N-Arylsulfonarnides 469
Androgenetic alopecia, 433,435, selective COX-2 inhibitors, Beriberi, 387-388,389
440
241-242 p,-Antagonists. See /3-Blockers
Angiogenesis
Ascorbic acid, 399. See also Vita- @Blockers
plasmid DNA-mediated gene
therapy, 656 min C gene expression profiling
Angiography, 567. See also Ra- Aspirin study, 612
diopaques discovery, 230 Bexarotene, 320,374
CT application, 568 effects on COX-1,244-245 Biguanides, 21-24
radiopaques applications, IL-12 inhibition, 180 BIIL 284,228,229
569-570 warning label, 427 Bioassays
Angiotensin-converting enzyme Astemizole peptide hormones, 52
(ACE) CYP3A41517 substrate, 627 Bioinformatics
single nucleotide polymor- removedlrestricted by FDA, DNA microarrays, 605-606
phims in insertionldeletion, 642 and gene expression profiling,
633 Asthma 614
Angiotensin, receptor and leukotrienes, 210 Biotin, 402-405
single nucleotide polymor- Atorvastatin deficiency, 405
phims, 633-635 P-glycoprotein inhibitor, 631 hypervitaminosis biotin, 405
Anthracyclines single nucleotide polymor- in leucine catabolism, 406
P-glycoprotein substrate, 631 p h i s m ~influencing response vitamin status assays, 367
Anthranilate sunscreens to, 644 Bis-ethylhexyloxyphenol me-
structure-activity relationship, thoxyphenol triazine .
Autoimunne diseases
469 (BEMT), 472
plasmid DNA-mediated gene
Antibody microarrays, 600 Bismuth subnitrate
therapy, 656 radiopaque, 489
Anticancer drugs
COX-2 selective inhibitors, Avandia, 25 Bis-triiodo-l,3-benezenedicar-
249-250 Avidin, 405 boxamide radiopaques, 497
lipiodol as carrier for, 494 Avobenzone Bis-triiodobenzoate radiopaques,
retinoids, 374 mechanism of action, 466 497
Anticancer gene therapy structure-activity relationship, Blockbuster drugs, 422,423
plasmid DNA-mediated, 653 469-470 B-lymphocytes. See B-cells
Antisense technology sunscreen, 460 BMS 180549
for gene expression profiling, radiopaque material, 491
613 Baby-boomer consumers, 423 Bordetellapertussis toxin
with plasmid DNA-mediated Baldness, 433. See also Hair effect on chemokines, 134
gene therapy, 666 growth disorder drugs BP-897
AOP-RANTES ligand Barium meals, 488 experimental smoking cessa-
and CCR1,137 Barium metatitanate tion agent, 459
and CCR3,150 radiopaque material, 493 BR2
and CCR5,155 Barium sulfate radiopaque material, 574
CCRl antagonist, 139 radiopaque, 487-489 BR21
Aortography, 567. See also Ra- Barium titanate radiopaque material, 574
diopaques radiopaque material, 488,493 Brain creatine kinase
Apex, 624 Basal cell carcinoma, 465 plasmid DNA-mediated gene
Aphrodyne, 442 Basen, 32 therapy study, 655
 Index
Breast cancer
cell characterization by gene
expression profiling, 608
tissue attenuation of x-rays,
486
Bremsstrahlung, 484
Bromfenac
removedlrestricted by FDA,
642
Brompheniramine
over-the-counter status, 426
Bronchography, 567. See also
Radiopaques
Bunamiodyl
biotransformation, 565
cholecystography application,
570
Bupropion, 451,452
adverse effects, 453
development of, 457-458
mechanism of action, 455
pharmacokinetics, 453-454
for sexual disorders, 450
structure-activity relationship,
456-457
B N ratio, of contrast agents,
538
BW-A4C, 213
BW-B70C, 213-214
BW-755C, 212
BX471, 142-143
Calcipotriene, 378
Calcitonin
and vitamin D, 376,377
Calcitriol, 377
Calcium
regulation by vitamin D fam-
ily, 376-377
Calcium pantothenate, 401,402
Calcium tungstate
radiopaque material, 493
Cancer
cell characterization by gene
expression profiling, 606-
608
and COX-2 selective inhibi-
tors, 249-250
Captopril
impact of ACE polymorphisms
on effectiveness, 634
Carbohydrate metabolism. See
also Glucose
enzymes involved in, 33-37
role of insulin, 2
Carboprost, 305
Carbutamide, 18
Carcinogenesis clinical efficacy and safety,
UV radiation effects, 465-466 248-249
Cardioangiography CYP2C9 substrate, 627
CT application, 568 in uiuo models, 246
Carisoprodol Cell-cell communication, 130-
CYP2C19 substrate, 627 131
a-Carotene, 369 Cell characterization
@-Carotene,320321,368- 370 by gene expression profiling,
asymmetrical cleavage, 322- 606-608
323 Cell surface receptors, 130
symmetrical cleavage, 321- Cellular retinol binding protein,
323,324
322
Central nervous system cancer
?-Carotene, 369
cell characterization by gene
Carotenes, 368 -371
expression profiling, 607,
Carotenoids, 320-321
608
Catagen phase, of hair growth, Cerivastatin
425 removed/restricted by FDA,
Caverjet, 442 642
CC1 receptor, 133,138-139 single nucleotide polymor-
antibodies, 139 p h i s m ~influencing response
peptide antagonists, 139 to, 644
small molecule antagonists, CGP-28238,242
139-143 CGS-25019c, 228,229
CC2 receptor, 133,143-144 Chemoinformatics
antibodies, 144 and bioinformatics, 614
peptide antagonists, 144-145 Chemokine modulators, 130-
small molecule antagonists, 131
145-148 Chemokine receptors, 130-131
CC3 receptor, 133,148-149 binding site model, 137
antibodies, 149 biology, 131-134
peptide antagonists, 149-150 CCR1,133, 138-143
small molecule antagonists, CCR2, 133,143-148
150-152 CCR3,133,148-152
CC4 receptor, 133, 152-153 CCR4,133,152-153
CC5 receptor, 133, 153-154 CCR5,133, 153-159
antibodies, 154-155 CCR6, 133,159-160
peptide antagonists, 155 CCR7,133,160-161
small molecule antagonists, CCR8, 133,161
155-159 CCR9,133
CC6 receptor, 133, 159-160 CCR10, 133
CC7 receptor, 133, 160-161 CC receptors, 132, 133, 136-
CC8 receptor, 133, 161 138
CC9 receptor, 133 CXCR1,161-165
CClO receptor, 133 CXCR2,161-165
CC receptors, 132,133,136-138 CXCR3,165-166
CD437,350 CX,CRl, 133
cDNA CXCR4,166-170
in plasmid DNA-mediated CXC receptors, 132, 133, 136-
gene therapy, 652 138
cDNA microarray chips, 604 ligand structure, 135
Celebrex, 204,233,234-235 model of, 132
clinical efficacy and safety, mutation and chimera studies,
248-249 137
Celecoxib, 234-235,239 sequence alignment of CC and
assays, selectivities, and po- CXC, 136
tencies, 245 signaling, 135

Chemokine receptors (Continued)
viral chemokines, 170-172
XCR1,133
Chemokines, 130-131
inducible vs. constitutive, 134
ChemR1,161
ChemR13,153
Chloramphenicol acetyltrans-
ferase, 27
Chlorpheniramine
over-the-counter status, 426
Chlorpropamide, 11, 12, 16
Cholangiography, 567. See also
Radiopaques
radiopaques applications,
569-570
Cholecalciferol, 374, 375. See
also Vitamin D,
keto and oxime analogs, 381
Cholecystography, 567. See also
Radiopaques
pharmacokinetics of ra-
diopaques, 559-562
radiopaques applications, 570
Chylomicrons
reconstituted fragments for
plasmid DNA-mediated
gene therapy, 667
CI-1004,246-247
Ciglitazone, 28
Cimetidine
classification in various coun-
tries, 430
Cinchophen
pretreatment effect on ra-
diopaques, 563
Cinnamate sunscreens
structure-activity relationship,
469
Cinoxate
structure-activity relationship,
469
sunscreen, 460
Cisapride
CYP3A4/5/7 substrate, 627
removedlrestrided by FDA, 642
Cisplatin
gene expression profiling
study of drug response, 609
P-glycoprotein substrate, 631
Citalopram
CYP2C19 substrate, 627
CKR-L1,161
CKR-L3,159
Clarithromycin
CYP3A41517 substrate, 627
P-glycoprotein inhibitor, 631
Clinical trials
gene expression profiling of
drug response subsets, 610
and pharmacogenomics, 641-
644
Clomipramine
CYP2C19 substrate, 627
Clonidine
yohimbine reverses ejacula-
tion inhibition, 446
Clotrimazole (vaginal)
classification in various coun-
tries, 430
Clustering methods
with DNA microarrays, 606
CMI 477
experimental smoking cessa-
tion agent, 459
Cobalamin, 405,408,411-415.
See also Vitamin B,,
Codeine
CYP2D6 substrate, 627
Codons, 619
Coenzyme A, 401
Cold medicines
OTC Drug Review, 426
Colon cancer
cell characterization by gene
expression profiling, 607,
608
COMPARE program, 609
Compton scattering, 485
Computer-assisted tomography
(CAT)
radiopaques applications, 495,
567-568
x-ray energies used, 484
Concerned member states, 429
Conjugase, 406
Constitutive chemokines, 134
Contrast agents. See Ra-
diopaques
Cortisol (hydrocortisone)
CYP3A41517 substrate, 627
over-the-counter status, 426
role in insulin regulation, 2
Corticosteroids
premedication for contrast
agents, 545
Cosmeceuticals, 423,473
Cotinine
experimental smoking cessa-
tion agent, 459
COX-1,205,230-232
role in prostaglandin synthe-
sis, 232-233
Index
COX-2,205,231-232
role in prostaglandin synthe-
sis, 232-233
COX-2 inhibitors, 204-205. See
also Leukotrienes
agents inhibiting both 5-10
and COX-2,246-248
assays, selectivities, and po-
tencies, 245-246
clinical efficacy and safety of
selective, 248-250
irreversible, 244-245
selective, 204,234-246
selective nonsteroidal anti-
inflammatory drugs, 242-
244
in vivo models, 246
CP 526555
experimental smoking cessa-
tion agent, 459
CS 891
experimental hair growth
drug, 441
CXCl receptor, 133, 161-162
antibodies, 162
peptide antagonists, 162-163
small molecule antagonists,
163-165
CXC2 receptor, 133, 161-162
antibodies, 162
peptide antagonists, 162-163
small molecule antagonists,
163-165
CXC3 receptor, 133,165-166
antibodies, 166
small molecule antagonists,
166
CX&1 receptor, 133
CXC4 receptor, 133,166-167
antibodies, 167
peptide antagonists, 167-168
small molecule antagonists,
168-170
CXC5 receptor, 133
CXC6 receptor, 133
CXC receptors, 132,133, 136-
138
Cyanocobalamin, 413
Cyclin-dependent kinase 2
gene expression profiling of
drug response, 611,612
Cyclin-dependent kinase cdc28p
gene expression profiling of
drug response, 611-612
Cyclooxygenase 112 inhibitors.
See COX-1 inhibitors;
COX-2 inhibitors
index
Cyclooxygenases, 229. See also
COX-1; COX-2; COX-1 in-
hibitors; COX-2 inhibitors
prostaglandin discovery and
NSAIDs, 229-230
Cyclosporine
CYP3A41517 substrate, 627
cytokine IL-1 downregulation,
173
cytokine IL-2 downregulation,
175
P-glycoprotein inhibitor, 631
P-glycoprotein substrate, 631
Cyclosporine A
cytokine IL-5 downregulation,
177
Cycloxygenase-derived eico-
sanoids, 233
Cystic fibrosis transmembrane
conductance regulator
plasmid DNA-mediated gene
therapy study, 664
Cytarabine
P-glycoprotein substrate, 631
Cytochrome P450
and leukotriene biosynthesis,
205,207
polymorphisms in CYP3A41
517, 626, 629-630
polymorphisms in CYP2C9,
626,628-629
polymorphisms in CYP2C19,
626,629
polymorphisms in CYP2D6,
626-628
Cytochrome P450 3A
polymorphisms in, 626, 629-
630
and vitamin D, 378
Cytokine modulators, 130-131,
172-173
IFNx 183-184
IL-1,173-174
IL-2, 174-175
IL-4,175-176
IG5,176-178
IL-6, 178-180
IL-12,180-181
IL-13,181-182
TNFa, 182-183
Cytokines, 130. See also specific
cytokines such as Erythro-
poietin
Cytokine syndrome, 175
Cytosine, 619
Cytotoxicity
plasmid DNA-mediated gene
therapy, 653,669-670
Dactinomycin
P-glycoprotein substrate, 631
Darbufelone, 246-247
Daunorubicin
P-glycoprotein substrate, 631
Debrisoquine, 627-628
hypotension from, 619
p Defensins
CCR6 binding, 159
7-Dehydrocholecalciferol,374,
375
7-Dehydrocholesterol, 374-375
Delta opioid receptor ligands,
69-75
Denaturing gradient gel electro-
phoresis, 622
Denaturing high performance
liquid chromatography
(dHPLC), 622
Dendritic cell-derived chemokine
(DCCK),150
Deoxynorjirimycin, 36
Desicocytes, 548
Desipramine
clinical trial using genotyping,
643
CYP2D6 substrate, 627
Dexamethasone
P-glycoprotein inducer, 631
P-glycoprotein substrate, 631
Dexfenfluramine
removedlrestricted by FDA,
642
Dextromethorphan
CYP2D6 substrate, 627
DFP, 240
DFU, 239-240
DHT. See 5a-Dihydrotestoster-
one
Diabeta, 13
Diabetes mellitus. See also Insu-
lin and hypoglycemic agents
type 1,2-4
type 2,2-4
Diabetic nephropathy, 3
Diabetic retinopathy, 3
Diabinese, 12
Diacylglycerol
elevation in hyperglycemia, 4
N-N-Diallylmelanine (DAM)
hair growth candidate drug,
438
Diamicron, 13
1,2-Diaminoyclohexane-N-,N'-
tetraacetic acid
radiopaque chelating agent,
489
Diatrizoate, 494,497, 536, 537
adverse reactions, 544
analysis, 539
angiography application, 569
biotransformation, 565,566
cardiovascular effects, 550,
551
cations used with, 541,542
excretion, 563,565
histamine release, 558-559
hyperosmolality, 542,543
liposome encapsulated, 573,
575
myelography application, 571
nephrotoxicity, 552
neurotoxicity, 553,554, 555
osmolality, 538,539
particulate, 576
pharmacokinetics, 561, 562
pharmacology, 540
properties, 504
protein binding, 556, 557
red blood cell effects, 549
spiral CT application, 569
urography application, 571
Diazepam
CYP2C19 substrate. 627 .
Dibenzoylmethane sunscreens
structure-activity relationship,
469-470
Diclofenac
classification in various coun-
tries, 430
Didesmethylsibutramine, 450
Dietary Reference Intakes, 361,
363-368
pantothenic acid, 400
vitamin A family, 371-372
vitamin B,, (cobalamin), 415
vitamin B, family, 400
vitamin B, (riboflavin), 392
vitamin B, (thiamine), 389-
392
vitamin D family, 379
vitamin E family, 384-385
vitamin K family, 387
Dietary supplements, 424
and future of over-the-counter
drugs, 473
increased popularity of, 422,
423

Dietary Supplements Education
and Health Act, 422
Diethanolamine methoxycin-
namate, 471-472
Diethylenetriaminepentaacetic
acid
radiopaque chelating agent,
489-490
Differin, 320
Differin Gel, 373
Diffuse androgen dependent alo-
pecia, 433
Digoxin
P-glycoprotein substrate, 631
Dihydropyrimidine dehydroge-
nase
gene expression effect on
5-fluoruracil,609
single nucleotide polymor-
phism~,630
5a-Dihydrotestosterone(DHT)
and hair growth, 435,436,439
Diiodophenyl alkanoate ra-
diopaques, 497
Diiodopyridone radiopaques, 497
Diltiazem
CYP3A41517 substrate, 627
P-glycoprotein inhibitor, 631
Dimethiodal sodium, 497
Dinoprost, 302
Dinoprosterone, 301302
Diodone
neurotoxicity, 555
Diodrast, 541
Dioxybenzone
structure-activity relationship,
469
sunscreen, 460
Diphenhydramine
over-the-counter status, 426
Diprotrizoate, 497
angiography application, 569
neurotoxicity, 554,555
properties, 504
Directive 92/26/EEC, 428
Direct-to-consumer advertising,
422
DNA, 619. See also Recombinant
DNA technology
DNA chips, 600
DNA insertion/deletion muta-
tions, 620
DNA microarrays
fabrication, 600-601
for gene expression analysis,
600-606
for genotyping, 622,624-625
interpretation and bioinfor-
matics, 605-606
DNA sequencers, 621
Docetaxel
P-glycoprotein substrate, 631
L-Dopa
interaction with vitamin B,
family, 399-400
Dose-response curves
vitamins, 362
Dovenex, 378
Doxercalciferol, 377, 378
Doxorubicin
P-glycoprotein substrate, 631
Drug characterization
DNA microarray application,
604
Drug Efficacy Study Implemen-
tation, 424
Drug interactions
vitamin B, family, 399-400
vitamin D family, 379-380
Drug metabolizing enzymes
polymorphisms in, 626-631
Drug target genes
polymorphisms in, 632-636
Drug transporter genes
polymorphisms in, 631-632
DRY6, 159
Duchenne muscular dystrophy
plasmid DNA-mediated gene
therapy study, 654,655
DuP 697,234,239
Durham-Humphrey amendment
(to Food and Drug Act), 424,
426
Dutasteride
experimental hair growth
drug, 440,441
Dyclonine
gene expression profiling
study, 613
Dymelor, 12
Early endosomes, 662
Echinocytes, 548
EDTA. See Ethylenediaminetet-
raacetic acid
Eflornithine, 433
adverse effects, 434,435
development of, 440
mechanism of action, 436-437
Eicosanoids, 204-205
biosynthesis, 205
cycloxygenase-derived, 233
Index
Electroporation
for plasmid DNA-mediated
gene therapy, 656-657
ELISA-type assays
for single base primer exten-
sion detection, 623
EM 250
experimental hair growth
drug, 441
Emulsions
iodinated suspension ra-
diopaques, 575-576
for plasmid DNA-mediated
gene therapy, 658,666-667
Enalapril
impact of ACE polymorphisms
on effectiveness, 634
ENA 78 ligand
and CXCR2,161
Enbrel, 182
Endosomal acidic insulinase, 8
England. See United Kingdom
Enkephalins
analogs leading to receptor-
specific ligands and nonpep-
tide ligands, 70-75
Enzyme-based genotyping tech-
nologies, 622-624
Eosinophils
chemokinelcytokine receptors,
133
Eotaxin
and CCR3,148-152
Eotaxin 1,149
.
Eotaxin 2,148,149
Eotaxin 3,148
Epidermal growth factor
plasmid DNA-mediated gene
therapy studies, 658,666
EPO. See Erythropoietin
Epoprostenol, 302-303
Epristeride, 438
Erectile dysfunction, 445-446,
448
Erections, 444-445
Ergocalciferol, 362,374,376
Erythrocytes
contrast media effects on,
547-550
Erythromycin
CYP3A41517 substrate, 627
P-glycoprotein inhibitor, 631
Erythropoietin, 130
Esophagography, 567. See also
Radiopaques
Essential amino acids, 362
Essential fatty acids, 362
Estimated Average Require- Fas ligand effect of dihydropyrimidine
ment, 363,368 plasmid DNA-mediated gene dehydrogenase expression
folic acid, 411 therapy study, 654 on, 609
niacin, 397 Fat soluble vitamins, 368 P-glycoprotein substrate, 631
vitamin A, 371 Fatty acids Fluoxetine
vitamin B,, 389 and insulin resistance, 28 CYP2D6 substrate, 627
vitamin B,, 392 role of insulin in metabolism, for premature ejaculation, 443
vitamin B,, 400 2-3 Flurbiprofen, 244
vitamin C, 418 FDA. See Food and Drug Admin- FMN, 393,394,397
vitamin E, 384 istration FMNH, 394
Estradiol Federal Food, Drug, and Cos- FMNH,, 394
CYP3A41517 substrate, 627 metics Act of 1938 Folic acid, 399, 405
Estrogens and over-the-counter drugs, and cobalamin, 415
for female unwanted hair 424 deficiency, 407-411
growth, 434 Felbamate Dietary Reference Intakes,
Etanercept, 182,183 removed/restricted by FDA, 401
Ethiodol 642 hypervitaminosis folic acid,
emulsified, 575 Female-pattern baldness, 433, 411
liposome encapsulated, 575 435 uptake and metabolism, 405-
radiopaque material, 493-494 Female sexual dysfunction, 440, 407
Ethyl diiodostearate 441-442 vitamin status assays, 367
radiopaque material, 493 Ferrites Food and Drug Administration
Ethylenediaminetetraacetic acid radiopaque, 487,489 (FDA)
(EDTA) radiopaque, ultrasmall, 487, over-the-counter drugs, 424-
radiopaque chelating agent, 490-492 428
489- 490 Fexofenadine sunscreen regulation history,
Ethyl triiodostearate P-glycoprotein substrate, 631 467,470-472
radiopaque material, 493 Fiber-optic biosensor arrays, 614 5-Formyl tetrahydrofolate poly-
Etodolac, 242-243 Fibrinogen glutamate, 407
Etoposide binding to contrast agents, 10-Formyltetrahydrofolate poly-
gene expression profiling 558 glutamate, 406,407,410
study of drug response, 609 Final monograph, over-the- Fosinopril
P-glycoprotein substrate, 631 counter drugs, 425-426 impact of ACE polymorphisms
Etoricoxib, 235,236 Finasteride, 433 on effectiveness, 634
Etrinate, 373-374 adverse effects, 434-435 FPL-55712,221
European Union development of, 439-440 FPL-55712-derived compounds,
over-the-counter classification mechanism of action, 436 221-222
in, 428-430 structure-activity relationship, FR-228352,235-236
Exons, 619 437-438 Fractalkine, 132
Expression analysis/profiling FK506 Fusin, 166
DNA microarrays for analysis, cytokine IL-1 downregulation,
600-606 173 Gadobenate dimeglumine
profiling applications in drug cytokine IL-2 downregulation, radiopaque material, 492
research, 606-614 173 Gadobutrol
cytokine IL-5 downregulation, radiopaque material, 492
Factor VII 177 red blood cell effects, 549
impact of polymorphisms on FK3311,241-242 Gadodiamide
acute coronary syndrome, Flosulide, 242 radiopaque material, 492
637,638 in vivo models, 246 Gadolinium chelates
FAD, 393,394,397 Flumizole, 234-235 radiopaque, 490-492,535
FADH, 394 Fluorescence resonance energy Gadolinium-DTPA
FADH,, 394 transfer (FRET) radiopaque material, 490,
Famotidine detection for genotyping, 622, 491-492
classification in various coun- 623,625 red blood cell effects, 549
tries, 430 5-Fluorouracil Gadopenamide
Farglitazar, 29-30 and DPD polymorphisms, 630 radiopaque material, 492

Gadopentetate dimeglumine
radiopaque material, 492
Gadoteric acid
radiopaque material, 492
Gadoteridol
radiopaque material, 492
Gadoversetamide
radiopaque material, 492
Gadoxetic acid
radiopaque material, 492
Gadozelite
radiopaque material, 492
Gastric acid secretion inhibitors,
86 -88
Ach-muscarinic-receptor-me-
diated secretion, 97
acid production, 88-90
CCK2/gastrin-receptorantago-
nists, 119
H. Pylori, 93-94
histamine H,-receptor-medi-
ated secretion, 97-103
hormones regulating, 89-90
physiology, 90 -91
proton-pump inhibition, 103-
117
receptor-mediated processing
regulating, 97-103
reflux esophagitis, 94-95
reversible proton-pump inhibi-
tors, 117-119
targets under investigation,
117-119
ulcer disease, 91-92
in vitro assays for, 95-96
in vivo assays for, 96-97
Zollinger-Ellison syndrome,
92-93
Gastritis, 86
Gastroesophageal reflux disease,
86
GCP-2 ligand
and CXCRl/CXCR2,161,162
Gemoprost, 303
Genechip, 624
Genechips
fabrication, 601
hybridization, 604-605
Gene deletions, 620
Gene duplication, 620
Gene expression
and drug response, 608-610
GeneFlex, 624
Gene function determination
DNA microarrays for, 604
Index
Gene gun cytokine IL-5 downregulation,
for plasmid DNA-mediated 177
gene therapy, 657-658 P-glycoprotein substrate, 631
Generally regarded as efficacious Gluconeogenesis
( G M ) , 424 insulin's role in regulating, 2
Generally regarded as safe Glucophage, 22
( G W ) , 424 Glucose. See also Diabetes melli-
Gene therapy, 652 tus; Insulin and hypoglyce-
Gene therapy, plasmid DNA- mic agents
mediated. See Plasmid normal range of plasma, 4
DNA-mediated gene therapy utilization by insulin, 2-4
Genetics, 618 Glucose toxicity, 3
Genome, 619 a-Glucosidase inhibitors, 3 1 3 7
Genomics a-Glucosidases, 33-37
and DNA microarrays, 600 Glucotrol, 13
and increased demand of con- Glutathione
sumers for control of their and vitamin E, 381,383
health, 422 Glyburide, 11, 13, 15, 16, 19,20,
simulating effects of drugs, 21,26
612-614 binding paramaters, 18
Genotype relative risk, 643 Glycogen
Genotyping, 600. See also Single role of insulin in synthesis, 2
nucleotide polymorphisms Glycogenolysis
combined enzymatichybrid- insulin's role in regulating, 2
ization approaches, 625-626 Glycoprotein IIIa
costs, 644 impact of polymorphisms on
hybridization-based, 624-625 acute coronary syndrome,
polymerase chain reaction- 637,638
restriction fragment length Glynase, 13
polymorphism, 622-623 Glyset, 32
pyrosequencing, 624 Gold
single base primer extension,
623-624
radiopaque material, 486 .
GPCRs (G-protein-coupledre-
use in clinical trials, 642-644
ceptors)
when to use, 621
prostanoid receptors as, 277-
Germany
over-the-counter classification, 278
428 GPCRs (G-protein-coupled re-
GG-745,440 ceptors), 7-transmembrane
GI-198745,440 and chemokilies, 131,132,135
Gliclazide, 13, 16 GPR-CY4,159
Glimepiride, 11, 13, 16,20 Grapafloxacin
binding paramaters, 18 removed/restricted by FDA,
CYP2C9 substrate, 627 642
Glinide insulinotropic agents, Great Britain. See United King-
11,14-20 dom
Glipizide, 11,13, 15, 16, 20 GROa ligand
binding paramaters, 18 and CXCR2,161
CYP2C9 substrate, 627 GROP ligand
Glucagon and CXCR2,161
role in insulin regulation, 2 GROy ligand
Glucagon-like polypeptide 1,34 and CXCR2,161
Glucoamylase-maltase, 33-36 Growth hormone
Glucocorticoids role in insulin regulation, 2
cytokine IL-1 downregulation, Guanine, 619
172,175 Gulonolactone oxidase, 417
Index
GW 320659
experimental smoking cessa-
tion agent, 459
GW 468816
experimental smoking cessa-
tion agent, 459
Habitrol, 452
Hair growth disorder drugs, 433
adverse effects, 434
chemistry and structure-activ-
ity relationship, 437-438
clinical use, 433-435
hair growth physiology, 435
history and future trends,
438-440
pharmacokinetics, 434-435
physiology and pharmacology,
435-437
role of 5-a-reductase, 435
Haplotypes, 620
Heavy metal radiopaques, 486-
493
Hectoral, 378
Helicobacter Pylori, 93-94
Hemagglutinins
plasmid DNA-mediated gene
therapy study, 657
Hemoglobin
glycosylated fraction as indica-
tor of glycemic control, 4
Hepatography, 567. See also Ra-
diopaques
Hepatolienography,567. See
also Radiopaques
Herpesviruses
chemokine receptors, 170-171
Hexobarbital
CYP2C19 substrate, 627
HHS-8 virus, 171
Higher osmolality intravascular
contrast agents, 495, 538
High performance liquid chro-
matography (HPLC)
denaturing, 622
High pressure liquid chromatog-
raphy (HPLC),48
Hirsutism, 433,440
H N fusion proteins
and CXCR4,166-170
HIV-1 virus
CCR5 and CXCR4 receptors,
154-155
HMG-CoA reductase inhibitors.
See Statins
Homing receptors. See Selectins
Homocysteine, 399, 408,412
Homosalate
structure-activity relationship,
468
sunscreen, 460
Hounsfield Unit, 567
Humalog, 7
Human Genome Project
and pharmacogenomics, 618,
619
Human genome sequencing, 618
and gene expression analysis,
600
Human serum albumin. See Se-
rum albumin
HUMSTR, 166
Humulin, 7
Hybridization-based genotyping,
624-625
Hydrocortisone. See Cortisol
Hydroperoxy-eicosatetraenoic
acid, 205
Hydroxy bupropion
experimental smoking cessa-
tion agent, 459
N-,N'-(2-Hydroxycyclohexy1)eth-
ylene-diaminediaceticacid
radiopaque chelating agent,
489
2-Hydroxycyclohexylethyl-
enediaminetriaceticacid
radiopaque chelating agent,
489
Hydroxy-eicosatetraenoic acid,
205
P-Hydroxyethylethylene diamine-
triacetic acid
radiopaque chelating agent,
489
Hydroxyhexamide, 16
Hydroxymethylglutaryl-CoA
(HMG-CoA)reductase in-
hibitors. See Statins
14-Hydroxy-4,14-retro-retinol,
327-328
N-Hydroxyureas
5-lipoxygenase inhibitors,
213-216
Hypercarotenosis, 371
Hypercholesterolemia
single gene pharmacogenetic
studies in genes influencing
progression, 639-641
Hyperglycemia, 2-4
Hyperinsulinemia, 3
Hypervitaminosis A, 319,370-
371
Hypervitaminosis biotin, 405
Hypervitaminosis C, 417
Hypervitaminosis D, 379
Hypervitaminosis E, 384
Hypervitaminosis folic acid, 411
Hypervitaminosis K, 387
Hypervitaminosis niacin, 395-
397
Hypervitaminosis pantothenic
acid, 401
Hypervitaminosis pyridoxine,
400
Hypervitaminosis riboflavin, 389
Hypervitaminosis thiamine, 389
Hypervitaminosis vitamin B,,,
415
Hypoglycemia, 2
side effect of insulin replace-
ment therapy, 5
Hypoglycemic agents. See Insu-
lin and hypoglycemic agents
Hysterosalpinography, 567. See
also Radiopaques
1-309,161
Ibuprofen
CYP2C9 substrate, 627
IC-351,450
ICI 198,615,225-226
ICI 204,219,225-226
Idiopathic hirsutism, 433,440 .
IES-40
radiopaque material, 577
IES-200
radiopaque material, 577
IFN-a. See Interferon a
IFN-y. See Interferon y
IL-la, 173
IL-lP, 173
Iletin 11, 7
Iloprost, 304305
IL-1R accessory protein, 173
Imidapril
impact of ACE polymorphisms
on effectiveness, 634
Imipramine
CYP2D6 substrate, 627
Imodium, 427
Impotency, 432,440-450
Incontinence, 432
Incretins, 34
Indinavir
CYP3A41517 substrate, 627
P-glycoprotein substrate, 631
Indolotropane,146

Indomethacin, 243
in vivo models, 246
Inducible chemokines, 134
Inflammation
animal models, 246
role of chemokines, 131-132
Infliximab, 182-183
Insulin
controlled substance, 423
Insulin, animal, 5
Insulin, [AspBlOI-,10-11
Insulin, genetically engineered,
5 Interleukin-6
Insulin analogs, 4-11
Insulin and hypoglycemic
agents, 1-11
biguanides, 21-24
formulations for subcutaneous
injection, 7
a-glucosidase inhibitors,
3 1-3 7
thiazolidinediones, 2 4 3 1
Insulin aspart, 6, 7,9
development of, 11
receptor binding affinities, 10
Insulin-degrading enzyme, 8
Insulin dependent diabetes mel-
litus. See Diabetes mellitus,
type 1
Insulin detemir, 11
Insulin glargine, 6, 7,9
development of, 11
receptor binding affinities, 10
Insulin lente, 6, 7
Insulin-like growth factor 1
insulin binding, 9-10
Insulin lispro, 6, 7, 9
development of, 11
receptor binding affinities, 10
Insulinotropic agents, 11-20
Insulin receptor, 9
Insulin resistance, 3
and free fatty acids, 28
Insulin-sensitizing agents,
20-31
Insulin ultralente, 6, 7, 9
Interdigitation-fusion vesicles,
573
Interferon y
IL-5 modulator, 177
modulator studies, 183-184
plasmid DNA-mediated gene
therapy study, 656,669-
670
Interleukin-1, 656
modulator studies, 173-174
Interleukin-2
glucocorticoid downregulation,
172
modulator studies, 174-175
Interleukin-4
modulator studies, 175-176
plasmid DNA-mediated gene
therapy study, 656
Interleukin-5
glucocorticoid downregulation,
172
modulator studies, 176-178
modulator studies, 178-180
plasmid DNA-mediated gene
therapy study, 657
Interleukin-8
and CXCRl/CXCR2,161-164
mutation and chimera studies,
137
Interleukin-10
plasmid DNA-mediated gene
therapy study, 656
Interleukin-12, 656
glucocorticoid downregulation,
17.2
modulator studies, 180-181
Interleukin-13
modulator studies, 181-182
Interleukins, 130
Internet
increased availability of medi-
cal information on, 423
Intrahepatic anion-binding Y
protein
binding to contrast agents,
557
Invader assay, 625
Invasive cleavage assays, 625
Investigational New Drugs Ap-
plication Process
over-the-counter drugs, 428
In vitro assays
gastric acid secretion inhibi-
tors, 95-96
In vivo assays
gastric acid secretion inhibi-
tors, 96-97
Iobenzamate
biotransformation, 566
Iobenzamic acid, 497
properties, 506
Iobitridol, 497
excretion, 564
nephrotoxicity, 552
properties, 506
Index
Iobutoic acid, 497
properties, 506
Iocarmate
excretion, 565
pharmacokinetics, 562
Iocarmic acid, 497
angiography application, 569
hyperosmolality, 543
myelography application, 571
properties, 508
synthesis, 514,515
Iocetamic acid, 497
cholecystography application,
570
excretion, 563
isomerism, 515
properties, 506
synthesis, 512,513
Iodamide, 497
angiography application, 569
excretion, 564
neurotoxicity, 554
pharmacokinetics, 561
properties, 504
urography application, 571
Iodamide meglumine, 497
Iodecol, 497
osmolality, 538,539
properties, 510
spiral CT application, 569
Iodinated particulate suspen-
sions
radiopaques, 572-577
.
Iodinated polymer radiopaques,
577
Iodine
x-ray cross-section, 486
Iodine organic radiopaque com-
pounds, 495-512
pharmacology, 540-566
potential nonionic, 518-535
structure-activity relationship,
517,531-539
synthesis, 512-517
Iodipamide, 497
adverse reactions, 544
biotransformation, 566
cholangiography application,
569-570
excretion, 562, 563
histamine release, 558
liposome encapsulated, 573
pharmacokinetics, 559-562
properties, 508
protein binding, 556-557
urography application, 571
1 Index
it Iodixanol, 497,538
J cardiovkcular effects, 551
cations used with, 542
development of, 495
excretion, 564
isomerism, 516
liposome encapsulated,
nephrotoxicity, 552
neurotoxicity, 554, 555, 556
osmolality, 539
pharmacokinetics, 562
properties, 510
protein binding, 556
red blood cell effects, 548,549
Iodized oil radiopaques, 493-495
Iodoalphionate
biotransformation, 565, 566
Iodoalphionic acid, 495,497
cholecystography application,
570
isomerism, 515
properties, 511
6-Iodoethylated starch, 577
Iodohippurate sodium, 497
urography application, 571
Iodomethamate
angiography application, 569
neurotoxicity, 555
urography application, 571
Iodophendylate, 497
properties, 511
Iodophenyl alkanoate ra-
diopaques, 497
Iodophthalein, 497
Iodopyracet, 497,541
angiography application, 569
biotransforrnation, 566
myelography application, 570
pharrnacokinetics, 561
properties, 511
protein binding, 557
urography application, 571
Iodoxamate
biotransforrnation, 566
pharmacokinetics, 559-561
Iodoxamate meglumine, 497
Iodoxamic acid, 497
cholangiography application,
570
excretion, 562,563
pharmacokinetics, 559
properties, 508
Iodoxyl, 497
properties, 511
Iofatrol
neurotoxicity, 555
Iofratol, 497
osmolality, 539
properties, 510
Ioglicic acid, 497
properties, 504
Ioglucol, 497
properties, 507
Ioglucomide, 497
properties, 507
Ioglunide, 497
neurotoxicity, 555
properties, 508
Ioglycamic acid, 497
biotransforrnation, 565
cholangiography application,
570
excretion, 562,563
properties, 509
protein binding, 556, 557
Ioglycamide
biotransforrnation, 566
cholangiography application,
570
histamine release, 559
pharmacokinetics, 559-560
Iogulamide, 497
properties, 508
Iohexol, 494,497,531
adverse reactions, 546
analysis, 539
angiography application, 569
cardiovascular effects, 550
cations used with, 541,542
development of, 495
excretion, 563-564
histamine release, 559
isomerism, 515
liposome encapsulated, 573
nephrotoxicity, 551, 552
neurotoxicity, 554, 555, 556
osmolality, 538,539
particulate, 576
pharmacokinetics, 561
pharmacology, 540
properties, 506
protein binding, 556, 557-558
red blood cell effects, 548,549
spiral CT application, 568
synthesis, 513,514
Iolidonic acid, 497
biotransforrnation, 565
properties, 505
Iolixanic acid, 497
properties, 505
Iomeglamic acid, 497
biotransforrnation,566
properties, 506
Iomeprol, 497
cardiovascular effects, 551
neurotoxicity, 554
osmolality, 538
properties, 506
red blood cell effects, 549
Smiles rearrangement, 513-
514
Iomorinic acid, 497
properties, 505
Iopamidol, 497,531,535
adverse reactions, 546
analysis, 539
angiography application, 569
biotransforrnation, 566
cardiovascular effects, 551
cations used with, 542
development of, 495
excretion, 564
histamine release, 559
isomerism, 515
liposome encapsulated, 573,
574
myelography application, 571
nephrotoxicity, 551,552
neurotoxicity, 554-555, 556
osmolality, 538
pharmacokinetics, 561
pharmacology, 540
properties, 506
protein binding, 556, 558
red blood cell effects, 548,549
Smiles rearrangement, 514
spiral CT application, 569
synthesis, 512, 513
urography application, 571
Iopanoate
biotransforrnation, 565-566
excretion, 562-563
liposome encapsulated, 575
pharmacokinetics, 561
Iopanoic acid, 497
adverse reactions, 544
biotransforrnation, 565, 566
cholecystography application,
570
excretion, 562, 563
isomerism, 515
properties, 505
protein binding, 556, 557
Iopax
properties, 511
urography application, 571
Iopentol, 497
analysis, 539
cardiovascular effects, 551
cations used with, 542

Iopentol (Continued) Iosumetric acid, 497
isomerism, 515 properties, 506
neurotoxicity, 554,555 Iotasul, 497
osmolality, 538 osmolality, 538
pharmacokinetics, 561-562 properties, 510
properties, 507 Iotetric acid, 497
protein binding, 557 properties, 509
red blood cell effects, 548, 549 Iothalamate
Iophendylate adverse reactions, 544
biotransformation, 566 angiography application, 569
excretion, 565 biotransformation, 566
myelography application, 570 cations used with, 541
Iophenoxate excretion, 562,563,565
biotransformation, 566 histamine release, 558,559
hyperosmolality, 542,543
Iophenoxic acid, 497
myelography application, 570-
biotransformation, 565-566
571
cholecystography application, neurotoxicity, 553-554, 555
570 pharmacokinetics, 561
excretion, 563 pharmacology, 540
isomerism, 515 protein binding, 556, 557
properties, 505 urography application, 571
protein binding, 556 Iothalamic acid, 497
3H2-Iopiperidol-A, 497 particulate, 576
properties, 507 properties, 505
Ioprocemic acid, 497 Iotranic acid, 497
properties, 505 properties, 509
Iopromide, 497 Iotriside, 497, 535
cardiovascular effects, 551 properties, 507
liposome encapsulated, 573- Iotrizoic acid, 497
575 properties, 504
nephrotoxicity, 551,552 Iotrol. See Iotrolan
neurotoxicity, 555 Iotrolan, 494,497,531
osmolality, 538 cardiovascular effects, 551
pharmacokinetics, 561 cations used with, 542
properties, 507 development of, 495
red blood cell effects, 549 histamine release, 558,559
Iopronic acid, 497 isomerism, 515
biotransformation, 565 liposome encapsulated, 573
properties, 506 nephrotoxicity, 551, 552
Iopydol, 497 neurotoxicity, 553, 554,555
properties, 511 osmolality, 537-538,538,539
Iopydone, 497 pharmacokinetics, 562
properties, 511 properties, 510
Iosarcol, 497 red blood cell effects, 549
properties, 508 Iotroxate
Iosefamic acid, 497 cholangiography application,
properties, 509 570
Ioseric acid, 497 Iotroxic acid, 497
properties, 505 biotransformation, 566
Iosimide, 497,535 pharmacokinetics, 559
analysis, 539 properties, 509
properties, 507 Ioversol, 497
Iosulamide, 497 adverse reactions, 546
cholangiography application, analysis, 539
570 cardiovascular effects, 551
properties, 509 excretion, 564
Index
isomerism, 515
liposome encapsulated, 573
nephrotoxicity, 551
neurotoxicity, 554, 555
osmolality, 538
pharmacokinetics, 561
properties, 507
red blood cell effects, 549
Smiles rearrangement, 514
synthesis, 512
Ioxabrolic acid, 497
properties, 510
Ioxaglate
adverse reactions, 546,547
cardiovascular effects, 551
cations used with, 542
histamine release, 559
hyperosmolality, 543
liposome encapsulated, 573,
574
nephrotoxicity, 551, 552
pharmacokinetics, 562
protein binding, 557
red blood cell effects, 548, 549
Ioxaglate meglumine, 494,497,
533
asymmetric bis compound,
537
development of, 495
osmolality, 538, 539
pharmacology, 540
synthesis, 514
Ioxaglic acid, 497
properties, 509
Ioxilan, 497
isomerism, 515
neurotoxicity, 554
particulate, 576-577
properties, 507
red blood cell effects, 548
synthesis, 513
Ioxitalamic acid, 497
adverse reactions, 546,547
angiography application, 569
cations used with, 541
properties, 505
red blood cell effects, 549
Ioxithalamate
histamine release, 558,559
neurotoxicity, 554
pharmacokinetics, 562
Ioxotrizoic acid, 497
properties, 504
Iozomic acid, 497
angiography application, 569
properties, 509
 Index

IP-10 ligand Keratin Leukocytes, 130

and CXCR3,165,166 and skin wrinkling, 373 chemokine-induced transmi-
Ipodate Ketoconazole gration, 131
biotransformation, 566 P-glycoprotein inhibitor, 631 Leukotriene 4
cholecystography application, Ketoprofen biosynthesis, 206-207
570 classification in various coun- discovery of, 208
excretion, 563 tries, 430 Leukotriene 4 hydrolase, 206,
Ipodic acid, 497 Knockout mice 207
properties, 505 for gene expression profiling broad distribution of, 208-209
Irbesartan studies, 613 Leukotriene 4 hydrolase inhibi-
CYP2C9 substrate, 627 IL-1,173 tors, 219-220
Ischemia. See Myocardial isch- IL-2, 175 Leukotriene antagonists
emia IL-4, 176 CystT1 antagonist clinical
Isofamic acid IL-5, 177 studies, 226-227
protein binding, 557 IL-6, 179 peptide-leukotriene antago-
Isomaltase-sucrase, 33-36 IL-12,180 nists, 220-226
Isomers IL-13,181 Leukotriene 4 synthase, 206
iodinated radiopaques, 515- INFy, 183 Leukotriene B,
516 TNFa, 182 biological actions of, 209
Isoniacid Kyoka (OTC license in Japan), biosynthesis, 206, 207
interaction with vitamin B, 430 discovery of, 208
family, 399,400,401 receptors, 209
Isoniazid Leukotriene B, antagonists
and NAT2 polymorphisms, L-674,636,217
derived from structure of LB,,
630 L-733692
228-229
peripheral neuropathy from, experimental hair growth
related to HAP-type LTD, an-
618 drug, 438,441 tagonists, 227-228
Isotretinoin, 320,373 L-745,337,242 Leukotriene biosynthesis inhibi-
I-TAC ligand L-761,000,243 tors, 210-211
and CXCR3,165 Labeling 5-lipoxygenase-activating pro-
Itraconazole over-the-counter drugs, 427- tein (FLAP) inhibitors,
P-glycoprotein inhibitor, 631 428 216-219
LabMAP, 624 5-lipoxygenase inhibitors,
Janus kinase, 130 Lactase, 33 211-216
chemokine interaction, 135 Lamotrigene LTA, inhibitors, 219-220
Japan clinical trial using genotyping, LTC, synthase, 220
over-the-counter classification 643 Leukotriene C,
in, 430-431 Lanosterol, 375 biological actions of, 210
Joduron, 541 Lansoprazole biosynthesis, 206, 207
JOM-13,70-71 CYP2C19 substrate, 627 discovery of, 208,209
JTE-522,239,240,241 Lanthanide chelates Leukotriene C, synthase, 206,
Justicidin E, 216 radiopaque materials, 490,491 207,209,220
Lantus, 7 Leukotriene D,
Latanoprost, 304 biological actions of, 210
Kallikrein-kinin system biosynthesis, 206, 207
plasmid DNA-mediated gene Lawsone, 471-472
discovery of, 208,209
therapy study, 654 Lead
Leukotriene D, antagonists,
KC 399 radiopaque material, 486 227-228
experimental hair growth Leflunamide, 175 Leukotriene E,
drug, 441 Lentiviruses biological actions of, 210
KC 516 chemokine receptors, 170-171 biosynthesis, 206, 207
experimental hair growth LESTR, 166 discovery of, 208,209
drug, 441 Leukemia Leukotriene F,, 207
Kefauver-Harris amendment (to cell characterization by gene Leukotriene modulators, 204-
Food and Drug Act), 424 expression profiling, 607, 205
Kelatorphan, 219-220 608 Leukotriene pathway, 206

Leukotrienes
biosynthesis, 205-208
discovery, 208
sources and actions of, 208-
210
LGD-1331
experimental hair growth
drug, 441
Life-enhancing drugs, 431
Lifestyle drugs, 422-423. See
also Over-the-counter drugs
current and future trends,
473-475
defined. 431
hair growth disorder drugs,
433-440
indications and drug exam-
ples, 432
and self-medication, 423
sexual disorder drugs, 440-
450
smoking cessation agents,
450-458
sunscreens, 458-472
Ligandin
binding to contrast agents,
557
Limaprost, 305
Linkage disequilibrium, 620
Lipid-based vectors
for plasmid DNA-mediated
gene therapy, 658-663
Lipids
protection from oxidation by
vitamin E, 383
Lipiodol
myelography application, 570
radiopaque material, 493,494
Lipolysis
insulin's role in regulating, 2
Lipoplex, 658
in vivo use, 663-665
Lipopolyplex, 658
for plasmid DNA-mediated
gene therapy, 665-666
Liposome-based plasmid DNA-
mediated gene therapy,
658-663
Lipoplex, 658,663-665
Lipopolyplex, 658,665-666
Liposomes
with gadolinium chelates as
radiopaque, 492
iodinated suspension ra-
diopaques, 572-575
5-Lipoxygenase
discovery of, 208
Index
in leukotriene biosynthesis, Lymphadenography, 567. See
205-207 also Radiopaques
single nucleotide polymor- Lymphangiography, 567. See
phism~,618 also Radiopaques
structure and mechanism, Lymphochip, 606
211-212 Lymphography, 567. See also
12-Lipoxygenase, 205 Radiopaques
15-Lipoxygenase, 205 Lymphokines, 130. See also Cy-
5-Lipoxygenase-activating pro- tokines
tein (FLAP) Lymphoma
in leukotriene biosynthesis, cell characterization by gene
205,206,207 expression profiling, 606
5-Lipoxygenase-activating pro- Lymphotactin, 132
tein (FLAP) inhibitors, 210, LY171883 (Tomelukast),221-
216-219 222
5-Lipoxygenase inhibitors, 210-
216 Magnesium ferrite
agents inhibiting both 5-LO radiopaque material, 489
and COX-2,246-248 Magnetic resonance imaging.
Lisinopril See Nuclear Magnetic Reso-
impact of ACE polymorphisms nance (NMR) imaging
on effectiveness, 634 Male-pattern baldness, 433,435
Lissoclin disulfoxide, 163, 164 Maltase-glucoamylase, 33-36
Lobeline, 457 MCP-1 ligand
Loperamide and CCR1,137-138
P-glycoprotein substrate, 631 and CCR2,143-147
Loratadine MCP-3 ligand
classification in various coun- and CCR1,138
tries, 430 and CCR3,148-149
Losartan MCP-4 ligand
CYP2C9 substrate, 627 and CCR3,148
P-glycoprotein substrate, 631 MCPd ligand .
Lovastatin and CCR2,144
gene expression profiling MCP-1R02 monoclonal antibody,
study, 613 144
Lower osmolality intravascular MCP-1RO5 monoclonal antibody,
contrast agents, 495,538 144
Lurnisterol, 374375 MDC ligand
Lung cancer and CCR4,153
cell characterization by gene Mecamylamine
expression profiling, 607, experimental smoking cessa-
608 tion agent, 459
LY-170680 (Sulukast), 222 Meglitinide, 20
LY-171883 (Tomelukast), 221- Meglumine salts, of contrast me-
222,226 dia, 541
LY-191704 Melanin
experimental hair growth and skin tanning, 464-465
drug, 437,441 Melanocortin receptors, 60
LY-255283,227 a-Melanocyte stimulating hor-
LY-293111,227-228 mone, 59-60
LY-354740 constrained cyclic lactam ana-
experimental smoking cessa- logs of agonists, 64-65
tion agent, 459 minimum active sequence,
LY-426965 60-61
experimental smoking cessa- P-Melanocyte stimulating hor-
tion agent, 459 mone, 59-60
Index

y-Melanocyte stimulating hor- 5-Methyltetrahydrofolatepoly- development of, 438-439

mone, 59-60 glutamate, 405,407,414 mechanism of action, 435-436
Melanogenesis, 464 Metoprolol structure-activity relationship,
Melanoma, 458,465-466 CYP2D6 substrate, 627 437
cell characterization by gene Met-RANTES ligand MIP-la ligand, 138-143
expression profiling, 607, and CCR1,139 antibodies to CCR1, 139
608 and CCR3,149-150 and CCR2,144
Melanotan 11,450 and CCR5, 155 and CCR5,153-155
Melanotropins Metrizamide, 497,531 MIP-1P ligand
antagonists, 65-67 development of, 495 and CCR5,153-155
constrained cyclic lactam ma- histamine release, 559 MIP-3a ligand
logs of a-MSH agonists, liposome encapsulated, 573 and CCR6,159
64-65 myelography application, 571 MIP-3P ligand
minimum active sequence of nephrotoxicity, 551,552
and CCR7,160-161
MSH, 60-61 neurotoxicity, 554,555, 556
Misoprostol, 303-304
D-Phe7 of MT-11: importance osmolality, 539
to binding and biological pharmacology, 540 Mitoxantrone
activity, 61-62 properties, 508 P-glycoprotein substrate, 631
ring-expanded analogs of MT- protein binding, 557 Mitrizamide
11, 63-64 red blood cell effects, 548 excretion, 565
structure-activity relationship, structure-activity relationship, pharmacokinetics, 562, 563
59- 60 517 MK-1,64-65
P-substituent effects, 62-63 Metrizoate, 497 MK-2, 66, 67
Meloxicam, 243 adverse reactions, 544 MK-3, 66, 67
Menadione, 385-386,387 angiography application, 569 MK-4, 66,67
Menoquinone-4,386 cations used with, 541,542 MK-434
Menthyl anthranilate excretion, 563 experimental hair growth
structure-activity relationship, hyperosmolality, 542,543 drug, 441
469 neurotoxicity, 554 MK-0476,223-224,226
sunscreen, 460 particulate, 576 MK-0571,223,226
Mephenytoin hydroxylase, 627 pharmacokinetics, 561 MK-0591,217,218
Metal chelates properties, 504 MK-0663,237
radiopaque, 489-490 red blood cell effects, 548 MK-0679,223,224,226 .
Metaproterenol sulfate inhaler urography application, 571 MK-0886
reversed from OTC back to Met-SDFlb, 155 FLAP inhibitor, 207,216-217
prescription status, 426 Met-SDF-1P ligand MK-966,235
Met-chemokine P-7, 150 and CXCR4,167 Mofetil, 175
Metformin, 21-23 Mexiletine Molluscum contagiosum virus
Methiodal sodium, 497 CYP2D6 substrate, 627 chemokine receptors, 171
Methotrexate MGSA ligand Monograph status, over-the-
folic acid with, 407,411 and CCR1,137-138 counter drugs, 425
with infliximab, 182-183 MH166 monoclonal antibody, 179 Monokines, 130. See also Che-
P-glycoprotein substrate, 631 Mibefradil mokines
Methoxsalen removed/restrictedby FDA, 642 Montelukast sodium (MK-04761,
experimental smoking cessa- Microelectroporation, 657 223-224,226
tion agent, 459 Micronase, 13 Moxisylyte, 442
a-Methylacarviosin, 36 Microsatellite nucleotide re- MR contrast agents, 567
P-Methylcrotonyl CoA carbox- peats, 620 MT-II,61-64, 66
ylase Midazolam Murine IL-4 mutants, 176
biotin dependent, 404 CYP3A41517 substrate, 627 Muse, 442
Methylene bis-benzotriazolyl MIG ligand Mutual Recognition Procedure
tetramethylbutylphenol and CXCR3,165,166 (European over-the-counter
(MBBT),472 Miglitol, 31, 32,33-34, 35, drugs), 428-429
5,lO-Methylenetetrahydrofor- 36-37 Myelography, 567. See also Ra-
mate polyglutamate, 406 Minoxidil, 433 diopaques
N-Methylglucamine, 541 adverse effects, 433 radiopaques applications,
4-Methylpyrogallol, 31 approval in Japan, 430 570-571

Myocardial diseases
plasmid DNA-mediated gene
therapy, 655-656
Myocardial ischemia
plasmid DNA-mediated gene
therapy study, 655
Nabi-NicVax
experimental smoking cessa-
tion agent, 459
NAD, 394,395,396
NADP, 394
Naked plasmid DNA
for plasmid DNA-mediated
gene therapy, 652-656
NAP-2 ligand
and CCR1,137-138
and CXCR2,161,162
Nateglinide, 11, 14, 15, 16, 17,
19,20
binding paramaters, 18
NCI 60 cells
characterization by gene ex-
pression profiling, 607-608
drug response screening by
gene expression profiling,
609
Negative-contrast radiocontrast
agents, 484
Negative monographs, over-the-
counter drugs, 425,426
Nelfinavir
and CXCR4,167
CYP3A4/5/7 substrate, 627
P-glycoprotein substrate, 631
Neotrophils
chemokinelcytokine receptors,
133
Neutral protarnine Hagedorn, 6,
7 Nitric oxide NMI-870, 450
New Drug Application
over-the-counter drugs, 424
New Jersey Genetic Privacy Act,
644
Niacin, 392-394
deficiency, 394-395
Dietary Reference Intakes,
397
and folic acid, 405
hypervitaminosis niacin, 395-
397
uptake and metabolism, 394
vitamin status assays, 366
Niacinamide, 394,396
Niacin equivalents, 361
Index
Nicorette, 452 Nonsteroidal anti-inflammatory
Nicotine drugs
peripheral pharmacological COX-2-selective, 242-244
actions, 455 development of, 230
pharmacological action, 454 and ulcers, 91-92
structure-activity relationship, Nonsynonymous single nucleo-
455 tide polymorphisms, 620
Nicotine dependence, 450 Northern blot hybridization
neurological basis of, 454-455 with DNA microarrays, 603-
Nicotine (gum), 451,452 604
adverse effects, 451 Nortriptyline
classification in various coun- CYP2D6 substrate, 627
tries, 430 Novolin, 7
development of, 457 Novolog, 7
pharmacokinetics, 453 NS-398, 241,242
Nicotine (patch), 451,452 NS-2359
adverse effects, 451 experimental smoking cessa-
classification in various coun- tion agent, 459
tries, 430 NSC65106,155
pharmacokinetics, 453 Nuclear Magnetic Resonance
Nicotine replacement therapy, (NMR)imaging
451 radiopaques for, 487,490-492
Nicotinic acetylcholine receptors Nuclear pore complex, 662
and smoking cessation agents, Nucleic acid vaccines, 657
455-457 Nucleus factor of activated T-
Nicotrol, 452 cells (NFAT), 173
Nicotrol inhaler, 452 Nulcear receptor boxes, 27
Nicotrol NS, 452 Nuvance, 176
Nifedipine
CYP3A4/5/7 substrate, 627 Obesity
NIH 10498 and insulin resistance, 3
experimental smoking cessa- Octocrylene
tion agent, 459 absorption and disposition, '
Nimesulide, 241 463
Nitric oxide sunscreen, 460
hyperglycemia effect on, 4 Octyl methoxycinnamate
reduction in hyperglycemia, 4 absorption and disposition,
role in constrast media toxic 462,463
effects, 549 structure-activity relationship,
role in erections, 445, 449 469
sunscreen, 460
Nitric oxide synthase Octyl salicylate
and erection, 445 absorption and disposition,
Nitric oxide synthase I1 463
plasmid DNA-mediated gene structure-activity relationship,
therapy study, 653 468
NitroMed, 450 sunscreen, 460
Non-insulin dependent diabetes Off-label use
mellitus. See Diabetes melli- lifestyle drugs, 431
tus, type 2 Oligonucleotide ligation assay,
Non-melanoma skin cancer, 465 625-626
Non-monograph status, over- Oligonucleotide microarrays,
the-counter drugs, 425 601-606
Non-peptide peptidomimetics, Omeprazole
72 CYP2C19 substrate, 627
Non-prescription drugs, 423,424 ON0 1078,224-225,226
ON0 4057,228,229
ON0 RS-411,224,226
ORF 74,171
Orinase, 12
Ornidyl, 440
Ornithine decarboxylase inhibi-
tors
for hair growth inhibition,
436-437
Osteoarthritis
COX-2 inhibitors for, 248-249
Osteoblasts, 376,377
Osteoclasts, 376, 377
Osteomalacia, 376
Osteoporosis, 376-377
Over-the-counter Drug Facts
label, 428, 429
Over-the-counter Drug Review,
424-426
classification after, 426-427
reclassification criteria for Rx-
OTC, 427-428
Over-the-counter drugs, 422-
423. See also Lifestyle drugs
classification in Europe, 428-
430
classification in Japan, 430-
431
classification in US., 424-428
current and future trends,
473-475
hair growth disorder drugs,
433-440
and self-medication, 423
sexual disorder drugs, 440-
450
smoking cessation agents,
450-458
sunscreens, 458-472
Oxybenzone
absorption and disposition,
462
sunscreen, 460
Oxytocin, 67
structure activity relationship,
67-68
Oxytocin antagonists, 66-69
P-1075
experimental hair growth
drug, 441
P53
and prostaglandin synthesis,
232
P509
radiopaque material, 578
Paclitaxel
P-glycoprotein substrate, 631
Padimate A, 468
Padimate 0
structure-activity relationship,
468
sunscreen, 460
Pancreatic p-cells, 2. See also
Insulin and hypoglycemic
agents
Pantoprazole
CYP2C19 substrate, 627
Pantothenic acid, 400-401, 403
Dietary Reference Intakes,
400
hypervitaminosis pantothenic
acid, 401
uptake and metabolism, 401
vitamin status assays, 367
Pantothenol, 401,402
Pantretin, 320, 374
Papaverine, 442,448
Parathyroid hormone
Alal and Val2 in signal trans-
duction, 55
ArgZ0and Arg5in receptor-
ligand interactions, 55
conformationally constrained
cyclic analogs of hPTH(1-
31)Amide, 58-59
Gly12in receptor-receptor in-
teractions, 55-56
structure-activity relationship,
53-55
truncated analogs: minimum
sequence requirements,
. 56-58
and vitamin D, 376
Parathyroid hormone-related
protein, 53-54
Parecoxib sodium, 241
Paricalcitol, 377, 378
Paroxetine
CYP2D6 substrate, 627
for premature ejaculation, 443
Particulates
iodinated suspension ra-
diopaques, 575-576
PC 1358
experimental hair growth
drug, 441
PD-138387,246-247
Pellagra, 392,394,395
Pelviagraphy, 567. See also Ra-
diopaques
Pentoxifylline
IL-12 inhibition, 180
Peptide hormones, 46- 48. See
also Arginine vasopressin;
Melanotropins; Oxytocin;
Parathyroid hormone
bioassays, 52
commonly held beliefs about,
47
conformation, 48-51
future directions, 75-76
synthesis, 48
topographical and conforma-
tional constraints, 52
topography, 51-52
Peptide-leukotriene antagonists,
220-221
diversified structures, 224-
226
FPL-55712-derived com-
pounds, 221-222
leukotriene analog antago-
nists, 222-224
Peptidomimetics, 72
future directions, 75-76
Perfluoroctyl bromide
radiopaque material, 571-572
Pernicious anemia, 407,415
Peroxisome proliferator-acti-
vated receptor a, 26,27
Peroxisome proliferator-acti-
vated receptor 6, 26,27
Peroxisome proliferator-acti-
vated receptor y, 26-27
Peroxisome proliferator-acti- .
vated receptor y agonists,
26-28
Peroxisome proliferator-acti-
vated receptor (PPAR) ago-
nists
thiazolidinediones, 24, 26-31
Petrolatum, 470
P-Glycoprotein efflux pump
polymorphisms in, 631-632
Pharmaceutical Affairs Law (Ja-
pan), 430
Pharmacogenetics, 619-620
Pharmacogenomics
combined enzymatickybrid-
ization genotyping, 625-626
enzyme-based genotyping
technologies, 622-624
genotyping technologies, 622-
625
hybridization-based genotyp-
ing, 624-625
influence on clinical trials,
641-644
obstacles facing, 644-646

Pharmacogenomics (Continued)
as paradigm shift in health
care, 645-646
polymerase chain reaction-
restriction fragment length
polymorphism genotyping,
622-623
polymorphisms in drug metab-
olizing enzymes, 626-631
polymorphisms in drug target
genes, 632-636
polymorphisms in drug trans-
porter genes, 631-632
pyrosequencing, 624
single base primer extension
genotyping, 623-624
single gene studies in genes
influencing disease progres-
sion, 636-641
SNP discovery, 621-626
Phenformin, 21,23
Phenidone, 212
Phenobarbital
interaction with vitamin D,
379-380
P-glycoprotein inducer, 631
pretreatment effect on ra-
diopaques, 563
Phentolamine,442,446,448
Phenylbenzimidazole sulfonic
acid
structure-activity relationship,
467
sunscreen, 460
Phenytoin
CYP2C9substrate, 627
P-glycoprotein inducer, 631
Phlebography, 567.See also Ra-
diopaques
CT application, 568
Phosphoinositol3 kinase y
chemokine interaction, 134,
135
Phospholipase C
role in chemokine modulation,
134
role in prostaglandin synthe-
sis, 233
Photoaging,465
Physical (particulate) sun-
screens
structure-activity relationship,
470
Phytonadione, 386,387
Pioglitazone, 24,25,28
binding affinities, 27
Plasma-DNA mediated gene
therapy, 651-652
Plasmid DNA-mediated gene
therapy
direct delivery with naked
plasmid DNA, 652-656
eledroporation, 656-657
emulsion-mediated gene
transfer, 666-667
entry of DNA into cells, 660-
662
entry of DNA into nucleus,
662-663
gene gun, 657-658
lipid-based vectors, 658-663
Lipoplex, 658,663-665
Lipopolyplex, 658,665-666
non-viral vector related cyto-
toxicity, 669-670
Polyplex, 658,667-669
Plasmids, 652,653
Platelet endothelial cell adhe-
sion molecule
plasmid DNA-mediated gene
therapy study, 669
Pobilukast (SKF 104353),222
Point mutations, 620
Polyamidoarnine dendrimers
for polyplexes, 668
Polyethyleneimines
for polyplexes,668
Polyiodinated triglycerides, 575-
576
Polymerase chain reaction, 621
Polvmerase
" chain reaction-re-
striction fragment length
polymorphism genotyping,
622-623
Polyplex, 658,667-669
Positive-contrast radiocontrast
agents, 484
Poxviruses
chemokine receptors, 170-171
Prandin, 14
Pranlukast (ON010781,224-
225,226
Pravastatin
single nucleotide polymor-
phism~influencing response
to, 639-641,644
p60 receptor, 182
p80 receptor, 182
Precose, 32
Pregnancy test kits, 424
Premature ejaculation, 440,441,
443
Prescription drugs, 423,424
Index
direct-to-consumer advertis-
ing, 422
reclassification to over-the-
counter status, 427-428
Prions, 46
Privacy concerns
genotyping, 644
Progesterone
CYP3A4/5/7substrate, 627
Promoter region, 619
Propafenone
CYP2D6 substrate, 627
Propecia, 433
Propionyl CoA carboxylase
biotin dependent, 404
Propranolol
CYP2D6 substrate, 627
Propyliodone, 497
properties, 511
Prostaglandin D, 230
Prostaglandin D,
physiological actions of, 271-
272
radioligand binding studies,
278
selective ligands and struc-
ture-activity relationhips,
282-285
signal transduction, 281
Prostaglandin E, 230
Prostaglandin E,-
physiological actions of, 272-
274
radioligand binding studies,
278-280
selective ligands and struc-
ture-activity relationhips,
285-289
signal transduction, 281
Prostaglandin FZa
physiological actions of, 274
radioligand binding studies,
280
selective ligands and struc-
ture-activity relationhips,
289-292
signal transduction, 281-282
Prostaglandin H synthase 1.See
COX-1
Prostaglandin H synthase 2.See
COX-2
Prostaglandin I,
physiological actions of, 274-
275
radioligand binding studies,
280
 Index
selective ligands and struc-
ture-activity relationhips,
292-294
signal transduction, 282
Prostaglandins
biosynthesis, 232-233
discovery and connection to
NSAIDs, 229-230
homeostatic effects, 233-234
and leukotriene biosynthesis,
205
pathophysiological effects, 234
role in erection, 445
Prostanoid receptors
classification and character-
ization, 269, 275-282
as G-protein-coupled recep-
tors, 277-278
ligand binding site loclization,
295-298
radioligand binding studies,
278-281
selective ligands and struc-
ture-activity relationhips,
282-295
signal transduction, 281-282
subtypes and isoforms, 276-
277
Prostanoids, 266
biological and therapeutic
functions, 267-269
biosynthesis of natural, 269-
270
clinical use of agents, 298305
physiological actions of, 271-
275
synthesis, 266-267
synthetic, 303-305
Protein hormones, 46-48
Protein kinase C
hyperglycemia effect on, 4
Protein kinase pathways
gene expression profiling stud-
ies, 610-611
Proteins
role of insulin in synthesis, 2
Prozac
lifestyle drug?, 431
Pseudoephedrine
over-the-counter status, 426
Psoriasis
retinoids used for, 373-374
PT-141,450
Pulmonary and activation-regu-
lated chemokine (PARC),
150
Pure Food and Drug Act of 1906, Rapamycin
424 cytokine IL-2 downregulation,
Pyelography, 567. See also Ra- 175
diopaques cytokine IL-5 downregulation,
Pyridoxal, 397 177
Pyridoxal phosphate, 397398, Rayleigh scattering, 485
399,400 Recombinant DNA technology
Pyridoxal phosphate biochemis- and plasmid DNA-mediated
try, 397-398 gene therapy, 652
Pyridoxamine, 397 Recommended Dietary Allow-
Pyridoxamine phosphate, 397 ances (RDA), 361,363-364,
Pyridoxine, 397,399,408 368
Pyridoxine phosphate, 397,399 folic acid, 411
Pyrosequencing, 624 niacin, 397
vitamin A, 372
Pyruvate carboxylase
vitamin B,, 389
biotin dependent, 404,405
vitamin B,, 392
vitamin B,, 400
Quality-of-life drugs, 431 vitamin C, 418
vitamin E, 384
Quinidine
P-glycoprotein inhibitor, 631 Recommended Nutrient Intakes,
363
Quinine
P-glycoprotein inhibitor, 631 Reconstituted chylomicron rem-
nants, 667
5a-Reductase
R-805,241 and hair loss, 435,439
Radiocontrast agents, 484,567 5a-Reductaseinhibitors, 426,
Radiolucent materials, 486 439-440
Radiopacity, 531-535 structure-activity relationship,
Radiopaque elements, 485 437-438
Radiopaque materials, 486 Reflux esophagitis, 94-95
Radiopaques, 483-486 Remicade, 182-183
applications, 566-571 Renal cancer
classification, 486 cell characterization by gene
heavy metals and their salts, expression profiling, 607,
486-493 608
iodized oils, 493-495 Renal failure
miscellanesous agents, 571- vitamin D analogs used in,
577 377-379
organic iodine compounds, Repaglinide, 11, 14, 15, 16-17,
495-566 19,20,21
potential nonionic, 518435 binding paramaters, 18
Ramipril Restriction fragment length
impact of ACE polymorphisms polymorphism genotyping,
on effectiveness, 634 622-623
Ranitidine Retin-A, 320,373
classification in various coun- Retinal, 319,362,368,369,370
tries, 430 conversion to retinol, 324,
FUNTES ligand 325-326
antibodies to CCR1, 139 Retinaldehyde, 323
and CCR1,137-138,142-143 Retinoic acid, 319,362, 368,
and CCR2,144 369370,374
and CCR3,148-150 active isomers, 326327
and CCR5,153-155 important signaling retinoid,
Rapacuronium 325-326
removedlrestricted by FDA, non-receptor binding proteins,
642 328

Retinoic acid (Continued)
RAR/RXR bind response ele-
ments, 339-340
signaling pathways, 335340
Retinoic acid-APL syndrome, 320
Retinoic acid receptor, 326
apo-receptors interact with
transcriptional corepressor
complexes, 341-344
binding domains, 340 3 4 1
bind response elements, 339-
340
holo complexes interact with
two classes of transcrip-
tional coactivators, 344-345
ligand binding domain, 341
ligand-independent transcrip-
tional activation, 345
Retinoids, 318-319. See also Vi-
tamin A family
anti-AP1 activity, 349-350
biosynthesis, 319-328
p-carotene asymmetrical
cleavage, 322323
p-carotene symmetrical cleav-
age, 321-322
CD437: atypical retinoid, 350
clinical applications, 318-319
and color vision, 334-335
fate of dietary sources, 320-
321
future directions, 345-351
gene-specific, 348-349
14-hydroxy-4,14-retro-retinol,
327-328
metabolism, 328330
receptor- and receptor sub-
type-selective, 345-348
role in vision transduction,
330335
used in acne treatment, 372373
used in malignancy treatment,
374
used in psoriasis treatment,
373374
Retinoid X receptor, 326
apo-receptors interact with
transcriptional corepressor
complexes, 341-344
binding domains, 340-341
bind response elements, 339-
340
holo complexes interact with
two classes of transcrip-
tional coactivators, 344-345
ligand binding domain, 341
Index
ligand-independent transcrip- CYP3A41517 substrate, 627
tional activation, 345 P-glycoprotein substrate, 631
novel uses for RXR-active SB 201993,228
compounds, 350351 SB 209247,228,229
Retinol, 368370. See also Vita- SB 240563,178
min A family SC-263,235,239
fate of intracellular, 324325 SC-588,232,235
oxidation, 325 SC-22716,219-220
Retinol binding protein, 324 SC-53228,227-228
Retinol dehydrogenases, 325 SC-57666,236,237
Retinyl esters SC-58125,235
biosynthesis, 323-324 SC-58451,235
Rev-5901,217 SC-65872,235
Reverse phase HPLC, 48 SC-574164 220
Rhodopsin, 324-325,362,368 SCH 54726
chemokine similarity, 135 experimental hair growth
cycle, 370, 371 drug, 441
Riboflavin, 392,393. See also Sch 55700, 178
Vitamin B, (Riboflavin) Schedule V Controlled Sub-
Riboflavin adenine dinucleotide, stances, 423
393 Scurvy, 417
Riboflavin phosphate, 392 SDF-la ligand
Rickets, 374,376 and CXCR4,166
Rifampin Secreted placental alkaline phos-
P-glycoprotein inducer, 631 phatase, 27
Ritonavir Selectan group, 495
CYP3A4/5/7 substrate, 627 Selectins
P-glycoprotein inhibitor, 631 and chemokine-induced trans-
P-glycoprotein substrate, 631 migration of leukocytes, 131
RNA polymerase, 619 Selenium
Robotics and vitamin E, 381
in DNA microarray fabrica- Self-medication, 423
tion, 601 Sequenom, 624 .
Roentgenography, 486 Serum albumin
Rofecoxib, 234-235 binding to contrast agents,
assays, selectivities, and po- 556-557,558
tencies, 245 Sexual disorder drugs, 440-442
clinical efficacy and safety, adverse effects, 443-444
248-249 chemistry and structure-activ-
in vivo models, 246 ity relationship, 446-447
Rogaine, 433 clinical use, 442-444
Rosiglitazone, 24, 25, 28, 30 erectile dysfunction causes,
binding affinities, 27 445-446
RP23618,139 erection physiology, 444-445
RU-58841 history and future trends,
experimental hair growth 447-450
drug, 441 pharmacokinetics, 444
physiology and pharmacology,
S-2474,247 444-446
Safety role of nitric oxide, 445
over-the-counter drugs, 427 Shonin (OTC regulatory ap-
St John's Wort proval in Japan), 430
P-glycoprotein inducer, 631 SHU9119,62
Salicylate sunscreens Sildenaiil (Viagra),440,442
structure-activity relationship, adverse effects, 443
468-469 CYP3A4/5/7 substrate, 627
Saquinavir development of, 448-449
Index
mechanism of action, 446
pharmacokinetics, 444
structure-activity relationship,
446-447
Simvastatin
single nucleotide polymor-
phism~influencing response
to, 639-641,644
Single base primer extension
genotyping, 623-624
Single nucleotide polymorphism
(SNP) maps, 620
Single nucleotide polymor-
phism~(SNPs),618-620.
See also Pharmacogenomics
classification, 620-621
defined, 620
discovery, 621-626
drug response and toxicity
variability, 618-619
frequency, 620
gene expression profiling of
drug response, 610
influence on clinical trials,
641-644
polymorphisms in drug metab-
olizing enzymes, 626-631
polymorphisms in drug target
genes, 632-636
polymorphisms in drug trans-
porter genes, 631-632
scanning for using DNA mi-
croarrays, 601
single gene studies in genes
influencing disease progres-
sion, 636-641
synonymous and nonsynony-
mous. 620
Single-strand conformation poly-
morphism analysis, 621
Singulair (MK-0476), 223-224,
226
Sirolimus
P-glycoprotein substrate, 631
SKF 104353 (Pobilukast),222,
226
SKF 106203,222
Skin cancer, 458
Skin tanning, 464-465
SLC ligand
and CCR7,160-161
Slow reacting substance of ana-
phylaxis (SRS-A),208
Smoking cessation agents, 450-
451
adverse effects, 451-453
chemistry and structure-activ-
ity relationship, 455-457
clinical use, 451-453
history and future trends,
457-458
nicotine's action, 454-455
pharmacokinetics, 453
physiology and pharmacology,
454-455
Snapshot, 624
SNPcode kits, 624
SNP Consortium, 622
SNPs. See Single nucleotide
polymorphisms
SNPStream 25K, 624
SNPware 96,624
SNuPe, 624
Sodium iodide
neurotoxicity, 555
Sodium iodomethamate, 497
properties, 511
Sodium ipodate
protein binding, 557
Sodium pantothenate, 401,402
Soft ferrites
radiopaque material, 489
Solange, 320
Solar keratosis, 466
Soriatene, 373
Sparteine hydroxylase, 627
Spin-lattice relaxation, 490
Spin-spin relaxation, 490
Spiral computer-assisted tomog-
raphy
radiopaques applications, 495,
568
Splenohepatography, 567. See
also Radiopaques
Splice site mutations, 620
Splicing, 619
Spotted DNA microarrays, 604-
605
Squamous cell carcinoma, 465
SR 141716
experimental smoking cessa-
tion agent, 459
SSR 591813
experimental smoking cessa-
tion agent, 459
Stanolone. See 5a-Dihydrotes-
tosterone
Starlix, 14
Statins
single nucleotide polymor-
phism~influencing response
to, 639-641,644
STRL-22,159
Succinylcholine
apnea from, 618-619
Sucrase-isomaltase, 33-36
4-Sulfonylphenyl COX-2 class,
234-241
Sulfonylurea insulinotropic
agents, 11-20
Sulisobenzone
absorption and disposition,
462,463
sunscreen, 460
Sulprostone, 304
Sulukast (LY170680), 222
Sunburn, 464
Sun protection factor, 467
Sunscreens, 458-460
absorption and disposition,
462-463
acute effects of ultraviolet ra-
diation, 464
adverse effects, 461-462
chemistry and structure-activ-
ity relationship, 467-470
chronic effects of ultraviolet
radiation, 464-466
clinical use, 460-463
history and future trends,
470-473
physiology and pharmacology,
463-467
protection factor, 467
Synonymous single nucleotide
polymorphisms, 620
T22
CXCFM antagonist, 167-168
TI40
CXCR4 antagonist, 168
Tachysterol, 375
Tacrolimus
CYP3A4/5/7 substrate, 627
P-glycoprotein inhibitor, 631
P-glycoprotein substrate, 631
TAK-779
CCR2 antagonist, 146-147
CCR5 antagonist, 156-157
Tamoxifen
P-glycoprotein inhibitor, 631
TA-NIC
experimental smoking cessa-
tion agent, 459
Tantalum
radiopaque material, 486-487
Tantalum oxide
radiopaque material, 492-493
Taqman assay, 625-626
TARC ligand
 Index

TARC ligand (Continued) plasmid DNA-mediated gene DNA microarray application,

and CCM, 153 therapy study, 668 604
Targretin, 320, 374 Thymine, 619 gene expression profiling ap-
Tat peptide Timolol plication, 614
CXCR4 antagonist, 168 CYP2D6 substrate, 627 Trace elements
Tazarotene Gel, 320,373,374 Titanium dioxide vitamins contrasted, 361-362
Tazorac, 320 absorption and disposition, Trail, 339
T-cell growth factor, 174 463 Transferrin
T-cells history of use as sunscreen, plasmid DNA-mediated gene
chemokine/cytokine receptors, 470 therapy study, 668
133 structure-activity relationship, Transgene
Tegison, 373 470 in plasmid DNA-mediated
Telogen phase, of hair growth, sunscreen, 460
gene therapy, 652
T-lymphocytes. See T-cells
425 Transgenic mice
TNFa converting enzyme
Tenidap, 179-180 IL-1,173
(TACE),182
Tentative final monographs, IL-2, 175
TNF a receptor. See also Tumor
over-the-counter drugs, 425 necrosis factor IL-4, 176
TER1,161 glucocorticoid downregulation, IL-5, 177
Terfenadine 172 IL-6, 179
removed/restricted by FDA, modulator studies, 182-183 IL-12,180
642 rac-a-Tocopherol, 381 IL-13,181
Testosterone a-Tocopherol, 380-384,399 INF-y, 183
CYP3A4/5/7 substrate, 627 Tocopherols, 380-384. See also TNFa, 182
and hair growth, 435,439 Vitamin E family Trazodone, 449-450
Testosterone patch, 450 Tocotrienols, 380381. See also CYP3A4/5/7substrate, 627
Tetraiodophenolphthalein, 495 Vitamin E family Tretinoin, 372373
cholecystography application, Tolazamide, 11, 12, 16 Triazolam
570 Tolbutamide, 11, 12,15 CYP3A4/5/7substrate, 627
Thiamine, 387-392. See also Vi- binding paramaters, 18 Tricomin
tamin B, CYP2C9 substrate, 627 experimental hair growth
Thiamine hydrochoride, 388 Tolerable upper intake level drug, 441
Thiamine kinase, 388-389 folic acid, 411 Triethanolamine salicylate
Thiamine nitrate, 388 niacin, 397 structure-activity relationship,
Thiamine pyrophosphate, 388, vitamin A, 371,372 468
389,390-391 vitamin B,, 400 sunscreen, 460
Thiazolidinediones, 24-31 vitamin C, 418 Triglycerides
Thiopurine methyltransferase vitamin D, 379 accumulation in hyperglyce-
single nucleotide polymor- vitamin E, 384-385 mia, 3,28
phism~,630-631 vitamins, 368 insulin's role in synthesis, 2
Thorium dioxide Tolinase, 12 Triiodoanilide radiopaques, 497
radiopaque material, 493 Tolterodine Triiodobenzamide radiopaques,
Thorium oxide CYP2D6 substrate, 627 497
neurotoxicity, 555 Tomelukast (LY171883),221- Triiodo-l,3-benzenedicarboxam-
Thorotrast, 493 222 ide radiopaques, 497
Thromboxane A,, 230 Tomexiprole, 242 2,4,6-Triiodobenzoate
physiological actions of, 275 Topoisomerase I1 inhibitors neurotoxicity, 554
radioligand binding studies, gene expression profiling Triiodobenzoate radiopaques,
280 study of drug response, 609 497
selective ligands and struc- Topotecan 2,3,5-Triiodobenzoicacid
ture-activity relationhips, P-glycoprotein substrate, 631 protein binding, 558
294-295 Torsemide Triiodoisophthalamate ra-
signal transduction, 282 CYP2C9 substrate, 627 diopaques, 497
Thromboxanes, 233 Toxicity Triiodophenoxy alkanoate ra-
and leukotriene biosynthesis, and single nucleotide polymor- diopaques, 497
205 phism~,618-619 Triiodophenyl alkanoate ra-
Thymidine kinase Toxicology diopaques, 497
Index
Triiodophenyl-D-gluconoamide
radiopaques, 497
Tris (2-amino-2-(hydroxy-
methyl)-1,3-propanediol),
541
Tris(iothalamic)acid, 497
properties, 511
Troglitazone, 24
binding affinities, 27
removedhestricted by FDA,
642
Trolamine salicylate
sunscreen, 460
Trovafloxacin
removed/restricted by FDA,
642
Tryptophan
niacin synthesis from, 361,
394,396
Tumor necrosis factor-a, 656.
See also TNFa receptor
plasmid DNA-mediated gene
therapy study, 670
Tumor necrosis factor-B
plasmid DNA-mediated gene
therapy study, 656
Tungsten
radiopaque material, 487
Turosteride
experimental hair growth
drug, 441
Type 1 diabetes mellitus. See
Diabetes mellitus, type 1
Type 2 diabetes mellitus. See
Diabetes mellitus, type 2
Tyropanoate, 570
biotransformation, 565
Tyropanoic acid, 497
properties, 505
UCB 35625,150
Ulcer disease, 91-92
Ultrasmall superparamagnetic
iron oxide particles, 491
Ultrasound contrast agents, 567
Ultraviolet radiation, 458-460
acute effects of, 464
chronic effects of, 464-466
United Kingdom
over-the-counter classification,
428
Urography, 567. See also Ra-
diopaques
pharmacokinetics of ra-
diopaques, 559-562
radiopaques applications, 571
Uroselectan
urography application, 571
Valdecoxib, 235
Validamine, 34-36
Valienamine, 34-36
Valiolamine, 34-36
Vaniqa, 433
Vanity drugs, 431
Vardenaiil, 450
Vascular endothelial growth fac-
tor
plasmid DNA-mediated gene
therapy study, 656
Venlafixine
CYP2D6 substrate, 627
Venography, 567. See also Ra-
diopaques
Ventriculography, 567. See also
Radiopaques
Verapamil
CYP3A4/5/7 substrate, 627
P-glycoprotein inhibitor, 631
Verlukast, 223,224
Vesanoid, 320
Viagra. See Sildenafil
Vinbcristine
P-glycoprotein substrate, 631
Vinblastine
P-glycoprotein substrate, 631
Vincristine
CYP3A4/5/7 substrate, 627
Vindesine
P-glycoprotein substrate, 631
Vinorelbine
P-glycoprotein substrate, 631
Vioxx, 204,233,234-235
clinical efficacy and safety,
248-249
Viral vectors
for gene therapy, 652
Viruses
chemokine receptors, 170-172
wectors for gene therapy, 652
Visual cascade, 325
Vital amine, 368
Vitamin A family, 368-369. See
also Retinoids
biochemical functions and de-
ficiency, 370
Dietary Reference Intakes,
371-372
dosage forms, 370
duel role of, 362
hypercarotenosis, 371
hypervitaminosis A, 319,370-
371
mobilization of body stores,
324
pharmacologically active reti-
noids, 372-374
storage of 6-9 month supply,
362
uptake and metabolism, 369-
370
vitamin status assays, 366
Vitamin B, (thiamine), 387-388
deficiencies, 389
Dietary Reference Intakes,
389-392
first vitamin whose structure
determined, 368
hypervitaminosis thiamine,
389
uptake and metabolism, 388-
389
Vitamin B, (Riboflavin),392
dosage and coenzyme forms,
393
Vitamin B, family, 397
deficiencies, 398-399
Dietary Reference Intakes,
400
drug interactions, 399-400
hypervitaminosis pyridoxine,
400
pyridoxal phosphate biochem-
istry, 397-398
uptake and metabolism, 397
Vitamin B,, (cobalamin),408,
411-413
deficiency, 415
Dietary Reference Intakes,
415
hypervitaminosis B,,, 415
recycling, 362
uptake and metabolism, 413-
415
Vitamin B12 (cobalamin)
vitamin status assays, 367
Vitamin B6 family
vitamin status assays, 367
Vitamin B2 (riboflavin)
vitamin status assays, 366
Vitamin B1 (thiamine)
vitamin status assays, 366
Vitamin C
vitamin status assays, 367
Vitamin C (ascorbic acid), 415
deficiency, 417
Dietary Reference Intakes,
418
hypervitaminosis C, 417

Vitamin C (ascorbic acid) (Con-
tinued)
uptake and metabolism, 415-
417
Vitamin D,, 374,376
not normal constituent of diet,
361
Vitamin D, (cholecalciferol),
374-380
not normal constituent of diet,
361
Vitamin D family, 374-375
biochemical function, 376-379
Dietary Reference Intakes, 379
dosage forms, 379
drug interactions, 379-380
hypervitaminosis D, 379
uptake and metabolism, 375-
376
vitamin status assays, 366
Vitamin D receptors, 376
Vitamin deficiencies, 362
biotin, 405
causes of, 362,364367
comparison of intake in areas
with/without deficiency dis-
ease, 363
folic acid, 407-411
vitamin A family, 370
vitamin B,, (cobalamin),415
vitamin B, family, 398-399
vitamin B, (riboflavin),392
vitamin B, (thiamine), 389
vitamin C, 417
vitamin D family, 376-379
vitamin E family, 383-384
vitamin K family, 386-387
Vitamin E family, 380381
biochemical function, 383
deficiencies, 383-384
Dietary Reference Intakes,
384-385
dosage forms, 384
hypervitaminosis E, 384
in sunscreens, 472
uptake and metabolism, 381-
383
vitamin status assays, 366
Index
Vitamin K1(20,,386 XC 1 receptor, 133
Vitamin K,, 386 Xenical, 474
Vitamin &,,,,, 386 X-ray contrast agents, 567
Vitamin K,, 385-386 X-rays, 484-486
Vitamin K base, 386
Vitamin K family, 385-386 Yeast genes
biochemical function and defi- with microarrays for expres-
ciency, 386387 sion profiling, 612
Yohimbine
Dietary Reference Intakes, 387
adverse effects, 443
hypervitaminosis K, 387
history of use, 447-448
uptake and metabolism, 386
mechanism of action, 446
vitamin status assays, 366
pharmacokinetics, 444
Vitamin &Hz, 386 structure-activity relationship,
Vitamin K oxide, 386 447
Vitamins Ytterbium-DTPA
adequate intake, 368 radiopaque material, 490
biochemical function satura-
tion, 363 Zafirlukast, 225-226
causes of deficiency, 362 Zaleplon
comparison of intake in areas CYP3A41517 substrate, 627
withtwithout deficiency dis- Zaprinast, 449
ease, 363 ZD2138,215-216
defined, 361 ZD4407,216
Dietary bference Intakes, Zein, 395
363368 Zemplar, 378
estimated average require- Zidovudine (AZT)
ment, 363 and CXCR4,167
fat and water soluble, 368 Zileuton
functions, 361-362 impact of 5-lipoxygenase poly-
human metabolic balance morphism~on effectiveness,
studies, 363 635
recommended dietary allow- Zileutron, 213-214 .
ances, 363368 Zinc oxide
serum levels, 363 absorption and disposition,
tolerable upper intake level, 368 463
valid dose determination, 362- history of use as sunscreen,
363 470
vitamin status assays, 366- structure-activity relationship,
367 470
VJH-085,66 sunscreen, 460
WL530,219 ZM211965,215
Voglibose, 32, 33, 34 Zollinger-Ellison syndrome, 86,
92-93
Warfarin Zolpidem
CYP2C9 substrate, 627 CYP3A41517 substrate, 627
Water soluble vitamins, 368 Zyban, 452
Wernicke-Korsakoffsyndrome, 389 Zyflo, 211,213-214,226-227
 work ... highly pr
5 studies and research.
dicinal Chem

This new edition of Dr. Alfred Burger's internationally celebrated

classic helps researchers acquaint themselves with both traditional
and state-of-the-art principles and practices governing new
medicinal drug research and development. Completely updated
and revised to reflect the many monumental changes that
have occurred in the field, this latest edition brings together
contributions by experts in a wide range of related fields to explore
recent advances in the understanding of the structural biology
of drug action, as well as cutting-edge technologies for drug
discovery now in use around the world.
This Sixth Edition of Burger's Medicinal Chemistry and Drug
Discovery has been expanded to six volumes:
Volume 1: Drug Discovery
Volume 2: Drug Discovery and Drug Development
Volume 3: Cardiovascular Agents and Endocrines
Volume 4: Autocoids, Diagnostics, and Drugs from New Biolo
Volume 5: Chemotherapeutic Agents
Volume 6: Nervous System Agents

DONALD A. ABRAHAM, PHD, is rrvlt 3r and C u a ~ r m

of the Department of Medicinal ChemisLL at the Virgir,,,
rnmmonwealth University School of Pharmacy. A world-renowned
ier in medicinal chemistry and biotechnology, he is the author
more than 140 journal citations and twenty-five patents. H e
s selected by the AACP Board of Directors as the recipient of the
02 AACP Paul Dawson Biotechnology Award.
 CAT

ocoids, Diagnostics, an
Drugs from New Biolog
 In addition to having cell numbers associ-
ated with this disease, the biology of the eosin-
ophil also implicates this cell as a major con-
tributor to the disease process. Eosinophils
are phagocytic granulocytes that mature in
bone marrow, migrate to the blood stream,
and eventually localize at the site of injury.
They contain highly toxic components that are
released upon degranulation, leading to de-
struction of bronchial epithelium, mucosal
edema, and bronchial hyperresponsiveness
IL-5 is detectable in bronchial biopsies and
bronchoalveolar lavage (BAL) fluid of asth-
matics (433, 434). Additionally, inhalation of
IL-5 by asthmatics has been shown to cause
airway hyperresponsiveness (AHR) and an in-
crease in sputum eosinophils (435).
IL-5 is produced primarily by activated
CD4t T-cells (436,437), and in lower levels by
eosinophils (438), mast cells (439, 4401, ba-
sophils, B-cells, NK cells (441,4421, and endo-
thelial cells (443). The expression of IL-5 is
regulated at the transcrip-
(444), and can be induced by a va-
ety of stimulants, usually through activation
11 receptor and a second signaling
thway. IL-la and PMA can induce IL-5 ex-
ine can also increase the pro-
L-5 in activated T-cells (445).
Structurally, IL-5 is a disulfide-linked ho-
modimeric glycoprotein between 45 and 60
kDa (446). Glycosylation does not seem to be a
requisite for signaling; however, IL-5 must be
in native dimeric form for bioactivity (447).
Each monomer of IL-5 consists of four a-heli-
ces with an antiparallel P-sheet between op-
posing monomers. The monomers are main-
tained in dimeric form by cysteine bonds at
residues 44 and 86 (448). Mutagenesis shows
residues His38, Lys39, and His41 in the sec-
ond helix; Glu89 and Arg91 in the p-strand;
and ThrlO9, GlullO, TRP111, and Is0112 in
the fourth helix as being important contribu-
tors to the IL-5 interaction with the hIL-5Ra-
chain (449,450). Glu13 on IL-5 has been iden-
tified as a contact point for the p-chain of the
a-chain specifically binds IL-5
with low affinity (451).When associated with a
@a]-transducing p-unit that is also used by
other hematopoietin receptors such as GM-
CSF and IL-3, IL-5 binds with high affinity
(452-454). Both subunits are necessary for
signal transduction (455). The pathway for
signal transduction includes activation of two
Janus kinases (JAK1 and JAK2) and the sig-
nal transduction/activator STAT5 (456,457).
9.4.1 11-5 Knockout and Transgenic Mice.
Animal models have helped define the signifi-
cance of IL-5 and the eosinophil in the disease
process. IL5 administered to mice results in an
increase of eosinophils (458). Experiments
with IL-5-deficient and IL-5-transgenic mice
confirm a role for this cytokine in controlling
eosinophilia (459,460). In IL-5 knockout mice,
no eosinophils are produced in response to
parasite infection or sensitization with
ovalbumin, and there is minimal development
of lung inflammation or tissue damage. When
IL-5 expression is reconstituted in these mice,
pulmonary eosinophilia, tissue destruction,
and airflow limitation can be observed after
allergen challenge (459).
Transgenic mice that have constitutive ex-
pression of IL-5 with detectable levels of IL-5
in the serum and persistent eosinophilia have
also been described (461). These mice are de-
scribed as normal, which may suggest that ac-
tivation and degranulation of eosinophils may .
be necessary for disease pathology.
9.4.2 11-5 Modulators/Clinical Data. Modu-
lation of IL-5 can occur by inhibiting its pro-
duction and synthesis, or through direct bind-
ing to IL-5 receptor or ligand. Cytokines can
regulate IL-5 levels by inhibiting production.
For example, IFNy and IL-10 have demon-
strated they can inhibit IL-5 production in
vitro (462), whereas IL-12 indirectly modu-
lates IL-5 by biasing toward a T h l subset pop-
ulation (463).
Small molecule antagonists such as CsA,
FK506, and rapamycin all inhibit IL-5 produc-
tion (464,465). Glucocorticoids, in addition to
decreasing bronchial hyperresponsiveness,
can also downregulate IL-5 production (466-
468). OM-01 suppresses IL-5 protein produc-
tion, mRNA expression, and transcriptional
activity in PBMCs with no effect on either IL-2
or IL-4 (469, 470). (The structure for OM-01
has not been disclosed.)